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Gambaryan A.S.,M P Chumakov Institute Of Poliomyelitis And Viral Encephalitides | Lomakina N.F.,Y R Kovalenko All Russian Research Institute Of Experimental Veterinary | Boravleva E.Y.,M P Chumakov Institute Of Poliomyelitis And Viral Encephalitides | Kropotkina E.A.,M P Chumakov Institute Of Poliomyelitis And Viral Encephalitides | And 4 more authors.
Influenza and other Respiratory Viruses | Year: 2012

Objective Parallel testing of inactivated (split and whole virion) and live vaccine was conducted to compare the immunogenicity and protective efficacy against homologous and heterosubtypic challenge by H5N1 highly pathogenic avian influenza virus. Method Four experimental live vaccines based on two H5N1 influenza virus strains were tested; two of them had hemagglutinin (HA) of A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site, and two others had hemagglutinins from attenuated H5N1 virus A/Chicken/Kurgan/3/05, with amino acid substitutions of Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2 while maintaining the polybasic HA cleavage site. The neuraminidase and non-glycoprotein genes of the experimental live vaccines were from H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/Gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). Inactivated H5N1 and H1N1 and live H1N1 vaccine were used for comparison. All vaccines were applied in a single dose. Safety, immunogenicity, and protectivity against the challenge with HPAI H5N1 virus A/Chicken/Kurgan/3/05 were estimated. Results All experimental live H5 vaccines tested were apathogenic as determined by weight loss and conferred more than 90% protection against lethal challenge with A/Chicken/Kurgan/3/05 infection. Inactivated H1N1 vaccine in mice offered no protection against challenge with H5N1 virus, while live cold-adapted H1N1 vaccine reduced the mortality near to zero level. Conclusions The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses. © 2011 Blackwell Publishing Ltd.


Sokolov A.V.,Saint Petersburg State University | Kostevich V.A.,Karpov Institute of Physical Chemistry | Zakharova E.T.,Research Institute of Experimental Medicine | Samygina V.R.,Russian Academy of Sciences | And 2 more authors.
Free Radical Research | Year: 2015

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latter's interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3′,5,5′-tetramethylbenzidine, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883-892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed. © 2015 Informa UK, Ltd.


Babich P.S.,Saint Petersburg State Polytechnic University | Skvortsov A.N.,Saint Petersburg State Polytechnic University | Rusconi P.,Mario Negri Institute for Pharmacological Research | Tsymbalenko N.V.,University of Helsinki | And 3 more authors.
Cancer Biology and Therapy | Year: 2013

To assess the statistical relationship between tumor growth and copper metabolism, we performed a meta-analysis of studies in which patients with neoplasms were characterized according to any of the copper status indexes (atomic copper serum concentration, serum oxidase activity, ceruloplasmin protein content). Our meta-analysis shows that in the majority of cases (more than 3,100 patients), tumor growth positively correlates with the copper status indexes. Nude athymic CD-1 nu/nu mice with subcutaneous tumors of human origin, C57Bl/6J mice with murine melanoma and ApcMin mice with spontaneously developing adenomas throughout the intestinal tract were studied to experimentally determine the relationship between tumor progression, liver copper metabolism, and copper status indexes. We showed that the copper status indexes increased significantly during tumor growth. In the liver tissue of tumor-bearing mice, ceruloplasmin gene expression, as well as the expression of genes related to ceruloplasmin metallation (CTR1 and ATP7B), increased significantly. Moreover, the presence of an mRNA splice variant encoding a form of ceruloplasmin anchored to the plasma membrane by glycosylphosphatidyl inositol, which is atypical for hepatocytes, was also detected. The ATP7A copper transporter gene, which is normally expressed in the liver only during embryonic copper metabolism, was also activated. Depletion of holo-ceruloplasmin resulted in retardation of human HCT116 colon carcinoma cell growth in nude mice and induced DNA fragmentation in tumor cells. In addition, the concentration of cytochrome c increased significantly in the cytosol, while decreasing in the mitochondria. We discuss a possible trans-effect of developing tumors on copper metabolism in the liver. © 2013 Landes Bioscience.


Ilyechova E.,Saint Petersburg State Polytechnic University | Skvortsov A.,Saint Petersburg State Polytechnic University | Zatulovsky E.,Saint Petersburg State Polytechnic University | Tsymbalenko N.,Research Institute of Experimental Medicine | And 3 more authors.
Journal of Trace Elements in Medicine and Biology | Year: 2011

There is an emerging link between copper metabolism, tumor growth and efficiency of antitumor treatment with platinum drugs or copper chelators. So there is an urgent need for well-defined and reproduced animal models with different states of copper metabolism. In the present study an animal model (rats and mice) with switching copper status in blood serum (copper concentration, oxidase activity and ceruloplasmin (Cp) protein content) is characterized. The drop of copper status is caused by addition of AgCl to fodder (Ag-animals). In rats and mice, the influence of silver ions on oxidase and ferroxidase activity of blood serum is similar, but copper concentration is reduced by 90% in rats, and by 60% in mice. The absorbed silver ions are transported to liver cells and included to Cp polypeptides, which are secreted to blood serum then. Cp, which circulates in bloodstream of Ag-animals contains silver atoms, and is misfolded, as judged by circular dichroism spectroscopy and differential scanning calorimetry. Single intraperitoneal or per oral injection of Cu(II) salt to Ag-animals causes recovery of oxidase and ferroxidase activity of blood serum within 4 hours in both rodent species, presumably by rapid metabolic insertion of copper into forming Cp in liver. The recovered copper status persists for 3 days under the continuing Ag-diet. The possibilities of use of Ag-rodents with switching copper status in investigation of influence of copper status on tissue-specific intracellular copper metabolism and role of copper in tumor genesis, bone metabolism and neurodegenerative diseases are discussed. © 2010 Elsevier GmbH.


Ovsyannikov V.I.,Research Institute of Experimental Medicine | Berezina T.P.,Research Institute of Experimental Medicine | Shemerovskii K.A.,Research Institute of Experimental Medicine
Bulletin of Experimental Biology and Medicine | Year: 2015

Inhibition of the contractile activity of the stomach induced by psychogenic stress persisted after blockade of muscarinic and nicotinic cholinergic receptors and α2 and β12-adrenergic receptors. Stress-induced increase in contractile activity in the proximal part of the duodenum persisted during blockade of muscarinic and nicotinic cholinergic receptors, β12-adrenergic receptors. At the same time, blockade of the above cholinergic and adrenergic receptors eliminated the stress-induced increase in contractive activity in the distal part of the duodenum. © 2015, Springer Science+Business Media New York.


Sokolov A.V.,Research Institute of Experimental Medicine | Golenkina E.A.,Moscow State University | Kostevich V.A.,Research Institute of Experimental Medicine | Vasilyev V.B.,Research Institute of Experimental Medicine | Sud'Ina G.F.,Moscow State University
Biochemistry (Moscow) | Year: 2010

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 μg/ml (normal CP concentration in plasma is about 300-400 μg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5-10 μg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2-10 μg/ml CP and decreased at doses of CP above 40 μg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed. © 2010 Pleiades Publishing, Ltd.


PubMed | Research Institute of Experimental Medicine
Type: Journal Article | Journal: Free radical research | Year: 2015

Myeloperoxidase (MPO) is a challenging molecular target which, if put under control, may allow regulating the development of inflammatory reactions associated with oxidative/halogenative stress. In this paper, a new kinetic method for assaying the halogenating activity of MPO is described. The method is based on measuring the rate of iodide-catalyzed oxidation of celestine blue B (CB) by oxygen and taurine N-chloramine (bromamine). The latter is produced in a reaction of taurine with HOCl (HOBr). CB is not a substrate for the peroxidase activity of MPO and does not react with hydrogen peroxide and superoxide anion radical. Taurine N-chloramine (bromamine) reacts with CB in molar ratio of 1:2. Using the new method, we studied the dependence of MPO activity on concentration of substrates and inhibitors. The specificity of MPO inhibition by non-proteolyzed ceruloplasmin is characterized. The inhibition of taurine N-chloramine production by neutrophils and HL-60 cells in the presence of MPO-affecting substances is demonstrated. The new method allows determining the kinetic parameters of MPO halogenating activity and studying its inhibition by various substances, as well as screening for potential inhibitors of the enzyme.


PubMed | Research Institute of Experimental Medicine
Type: Comparative Study | Journal: Free radical research | Year: 2015

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latters interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3,5,5-tetramethylbenzidine, and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883-892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.


PubMed | Research Institute of Experimental Medicine
Type: Journal Article | Journal: Metallomics : integrated biometal science | Year: 2014

The influence of short and prolonged diet containing silver ions (Ag-diet) on copper metabolism was studied. Two groups of animals were used: one group of adult rats received a Ag-diet for one month (Ag-A1) and another group received a Ag-diet for 6 months from birth (Ag-N6). In Ag-A1 rats, the Ag-diet caused a dramatic decrease of copper status indexes that was manifested as ceruloplasmin-associated copper deficiency. In Ag-N6 rats, copper status indexes decreased only 2-fold as compared to control rats. In rats of both groups, silver entered the bloodstream and accumulated in the liver. Silver was incorporated into ceruloplasmin (Cp), but not SOD1. In the liver, a prolonged Ag-diet caused a decrease of the expression level of genes, associated with copper metabolism. Comparative spectrophotometric analysis of partially purified Cp fractions has shown that Cp from Ag-N6 rats was closer to holo-Cp by specific enzymatic activities and tertiary structure than Cp from Ag-A1 rats. However, Cp of Ag-N6 differs from control holo-Cp and Cp of Ag-A1 in its affinity to DEAE-Sepharose and in its binding properties to lectins. In the bloodstream of Ag-N6, two Cp forms are present as shown in pulse-experiments on rats with the liver isolated from circulation. One of the Cp isoforms is of hepatic origin, and the other is of extrahepatic origin; the latter is characterized by a faster rate of secretion than hepatic Cp. These data allowed us to suggest that the disturbance of holo-Cp formation in the liver was compensated by induction of extrahepatic Cp synthesis. The possible biological importance of these effects is discussed.


PubMed | Research Institute of Experimental Medicine
Type: Journal Article | Journal: Bulletin of experimental biology and medicine | Year: 2015

Inhibition of the contractile activity of the stomach induced by psychogenic stress persisted after blockade of muscarinic and nicotinic cholinergic receptors and 2 and 1/2-adrenergic receptors. Stress-induced increase in contractile activity in the proximal part of the duodenum persisted during blockade of muscarinic and nicotinic cholinergic receptors, 1/2-adrenergic receptors. At the same time, blockade of the above cholinergic and adrenergic receptors eliminated the stress-induced increase in contractive activity in the distal part of the duodenum.

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