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Saint Petersburg, Russia

Prokofieva D.S.,Research Institute of Hygiene | Goncharov N.V.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry
Tsitologiya | Year: 2014

Effects of biogenic and abiogenic disulphides on viability of human umbilical vein endothelial cells in culture has been investigated using three methods: the neutral red uptake assay, quantification of intracellular ATP, and modifications of Mosmann method, the essence of which is the reduction of tetrazolium salts, MTT and MTS, by cells. 2,2'-dithio-bis(N,N-diethyl)ethanamine (DS) was used as an abiogenic disulphide. As for biogenic disulphides, we used GSSG and garlic oil (GO), the principal component of which is diallyl disulphide (DADS). It has been found that DS and GO have a similar cytotoxic effect upon the endothelial cells (EC50 ~ 0.6 mM). GSSG in concentrations up to 1 mM did not effect the viability of endothelial cells. It has been demonstrated for the first time that DS and GO can serve as mediators of plasma membrane oxidoreductase activity, tetrazolium salts being as the substrate; this may cause false-negative effect. Thus, the Mosmann method has serious limitations when testing the cytotoxicity of disulphides, though can be used in studying the mechanism of action of disulphides. Source


Prokofieva D.S.,Research Institute of Hygiene | Goncharov N.G.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry
Cell and Tissue Biology | Year: 2014

Effects of biogenic and abiogenic disulfides on viability of human umbilical vein endothelial cells in culture were studied using three methods: inclusion of neutral red (NR) into cells, quantification of intracellular ATP, and modifications of the Mosmann method (1983), the essence of which consists in reduction of tetrazolim salts—MTT and MTS—by cells (Mosmann, 1983). 2,2′-Dithio-bis(N,N-diethyl)dietanamine (DS) was used as an abiogenic sulfide. As for biogenic disulfides, we used oxydized glutathione (GSSG) and garlic oil (GO), the major component of which is diallyl disulfide (DADS). It has been established that DS and GO have a compatible cytotoxic effect (ES50amounts to ∼0.6 mM), whereas GSSG at a concentration up to 1 mM had no effect on the viability of endothelial cells. It has been demonstrated for the first time that DS and GO can serve as mediators of plasma-membrane oxidoreductase activity, with tetrazolium salts being used as the substrate, which may cause a false-negative effect. In this connection, Mosmann’s method has serious limitations in evaluating the effect of disulfides on cell viability, although it can be used when studying the mechanism of their action. © 2014, Pleiades Publishing, Ltd. Source


Prokofieva D.S.,Research Institute of Hygiene | Jenkins R.O.,De Montfort University | Goncharov N.V.,Research Institute of Hygiene
Analytical Biochemistry | Year: 2012

Two microplate spectroscopic methods for determination of organophosphates, based on inhibition of acetylcholinesterase (AChE) activity, were further improved and evaluated for determination of the chemical weapon agent Russian VX (RVX) in aqueous solutions. The linear range of the Hestrin method (74.8-1120 pM) was 3.1-fold wider than that of the Ellman method (37.4-374 pM). Limits of detection and quantification of RVX for both methods were below the maximal allowable concentration of RVX in water-soluble washouts. One of the early products of RVX hydrolysis, N,N-diethylaminoethanethiol, like reduced glutathione, caused false negative results in the Ellman method at concentrations exceeding 10 μM; individual blanks were necessary to eliminate the effect. The Hestrin method showed greater specificity (∼3 orders of magnitude) for analysis of samples containing mercaptans. A major product of RVX degradation, 2,2′-dithiobis(N,N-diethylethanamine), caused significant inhibition of AChE at concentrations of ≥0.1 mM (P < 0.01) and had a false positive effect at higher concentrations (≥2 mM). For environmental monitoring of RVX, the method based on Hestrin is preferred over that based on Ellman, principally because the former method was less sensitive to interference from major admixtures and did not give rise to potentially dangerous false negative results. © 2011 Elsevier Inc. All rights reserved. Source


Prokofieva D.S.,Research Institute of Hygiene | Voitenko N.G.,Research Institute of Hygiene | Gustyleva L.K.,Research Institute of Hygiene | Babakov V.N.,Research Institute of Hygiene | And 3 more authors.
Journal of Environmental Monitoring | Year: 2010

Two microplate spectroscopic methods for determination of organophosphates, based on inhibition of acetylcholinesterase activity, have been elaborated and evaluated for determination of the chemical weapon agent soman. The principal difference between the methods is that one measures reaction substrate concentration (elaborated from Hestrin), while the other measures reaction product (elaborated from Ellman). The linear ranges of the two methods were found to be similar. Although the limit of quantification was lower for the Ellman method (110 pM), the sensitivity coefficient was in favor of the Hestrin method (1.55-fold higher). The effects of the main soman hydrolysis products were consistent for the two methods: both methylphosphonic acid and pinacolyl methylphosphonic acid did not inhibit acetylcholinesterase activity. The main components of decontaminating solutions showed differential effects: while monoethanolamine had no influence upon results obtained by either method, hydrogen peroxide interfered with the Ellman method at far lower concentrations than with the Hestrin method. In practical applications involving samples containing hydrogen peroxide, the method based on Hestrin should be regarded as much more specific for OP determination than the Ellman method. © 2010 The Royal Society of Chemistry. Source


Goncharov N.V.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry | Belinskaya D.A.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry | Razygraev A.V.,Do Ott Research Institute Of Obstetrics And Gynecology | Ukolov A.I.,Research Institute of Hygiene
Russian Journal of Bioorganic Chemistry | Year: 2015

Albumin molecules, unlike molecules of many other plasma proteins, are not covered with a carbohydrate shell. Albumin plays a crucial role in maintaining colloid osmotic blood pressure and can bind and transport various endogenous and exogenous molecules. The enzymatic activity of albumin, the existence and the role of which most researchers are still skeptical to accept, is our main interest. In this review, a history of the issue is traced, with particular attention paid to the esterase activity of albumin. The kinetic and thermodynamic characteristics of the interaction of albumin with some substrates are adduced, and the possibility of attributing albumin to certain groups of Enzyme Nomenclature is considered. © 2015 Pleiades Publishing, Ltd. Source

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