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Chernyavsky A.I.,University of California at Irvine | Arredondo J.,University of California at Irvine | Skok M.,Palladin Institute of Biochemistry | Grando S.A.,University of California at Irvine
International Immunopharmacology | Year: 2010

Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a 'cholinergic (nicotinic) anti-inflammatory pathway'. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide up-regulated expression of α1, α4, α5, α7, α10, β1 and β3 subunits, down-regulated α6 and β2 subunits, and did not alter the relative quantity of α9 and β4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro- and anti-inflammatory cytokines. While inhibition of the expression of the TNF-α gene was mediated predominantly by the α-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes-by the mecamylamine-sensitive nAChRs. Both the Mec- and αBtx-sensitive nAChRs regulated expression of the IL-1β gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through α7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists. © 2009 Elsevier B.V. All rights reserved. Source

Demchenko A.P.,Palladin Institute of Biochemistry
Experimental Oncology | Year: 2012

The strong plasma membrane asymmetry existing in living cells is lost on apoptosis, and it is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the labeled annexin V is used for its detection. The requirement for early and Ca2+-independent detection of apoptosis in the formats of spectroscopy of cell suspensions, flow cytometry, microarray technology and confocal or two-photon microscopy stimulated efforts for the development of new methods. Since the PS exposure must produce integrated changes of electrostatic potential and hydration in the outer leaflet of cell membrane, its detection can be provided by direct response of smart fluorescence probes. This review is focused on basic mechanisms underlying the loss of membrane asymmetry during apoptosis and the principles lying in the background of new methods that demonstrate essential advantages over the annexin V-binding assay. The convenient wavelength-ratiometric technique based on fluorescent probe F2N12S is described in detail. It incorporates spontaneously into outer leaflet of cell membrane and the color change of its fluorescent emission associated with apoptosis can be easily detected. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later". Copyright © Experimental Oncology, 2012. Source

Chernyshenko V.O.,Palladin Institute of Biochemistry
Protein Journal | Year: 2015

Previously we purified fibrinogenase from venom of Echis multisquamatis and showed that the enzyme predominantly cleaves BβArg42-Ala43 peptide bond of fibrinogen. A much slower hydrolysis of its Aα-chain was also shown. To evaluate the accessibility of the hydrolysis sites to fibrinogenase’s hydrolytic action, the pathway of cleavage of Aα- and Bβ-chains of fibrinogen, monomeric and polymeric fibrin desA and desAB has been investigated using western blot with monoclonal antibodies to Bβ 26–42 and Aα 20–78 of fibrinogen. The data indicated that the BβArg42-Ala43 peptide bond is available for cleavage in all forms of fibrin(ogen) with the exception of polymerized fibrin desAB. This is direct evidence of BβN-domain involvement in formation of protofibrils that makes it inaccessible to protease. The Aα-chain of fibrinogen remained intact after 3 min of incubation with fibrinogenase. Further incubation resulted in cleaving of the fibrin(ogen) αC-regions with the formation of two kinds of degradation products (~30 and ~60 kDa). In the case of monomeric fibrin desA or desAB we observed simultaneous hydrolysis of Aα and Bβ-chains and the cleavage of Aα-chain was more apparent for both forms of polymeric fibrin. © 2015, Springer Science+Business Media New York. Source

Demchenko A.P.,Ukrainian Academy of Sciences | Demchenko A.P.,Palladin Institute of Biochemistry
Journal of Fluorescence | Year: 2010

Very limited number of parameters is available for fluorescence sensing and imaging. The changes of intensity are of low analytical value due to the absence of internal reference. Anisotropy and lifetime sensing have their own limitations. In this respect the λ-ratiometric (based on intensity ratios at two or more wavelengths) recording of spectral changes becomes more popular. Because the spectral changes are connected directly with the variations of interaction energies this approach is seen as the most universal method to study intermolecular interactions. It is applicable for different sensor formats and for obtaining analytical information from cell images. Here we critically analyze different approaches in λratiometric sensing that use single and double fluorescence emitters and are based on different mechanisms producing spectroscopic change. Very promising is the exploration of mechanisms that allow obtaining ratiometric response from a single dye. © Springer Science+Business Media, LLC 2010. Source

Agency: Cordis | Branch: FP7 | Program: CSA-CA | Phase: SiS-2008- | Award Amount: 1.08M | Year: 2009

ETHICAL project has a set of concrete objectives to fulfil: 1. To formulate an international dialogue on ethical implications of data collection, use and retention in medical and biometric applications, in three specific themes: potential data misuse, development of a unique identifier and international standardisation of ethical requirements 2. To develop a guide on government industry collaboration prerequisites concerning the data collection, use and retention in medical and biometric applications. 3. To develop a code of conduct for FP7 researchers, concerning the data collection, use and retention in medical and biometric applications. 4. To identify the set of ethical requirements for international biometric and medical data sharing. 5. To create synergies with SINAPSE e-community of National Ethics Councils.

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