Palladin Institute of Biochemistry

Kiev, Ukraine

Palladin Institute of Biochemistry

Kiev, Ukraine
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Lykhmus O.,Palladin Institute of Biochemistry | Gergalova G.,Palladin Institute of Biochemistry | Koval L.,Palladin Institute of Biochemistry | Zhmak M.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | And 2 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2014

Nicotinic acetylcholine receptors control survival, proliferation and cytokine release in non-excitable cells. Previously we reported that α7 nicotinic receptors were present in the outer membranes of mouse liver mitochondria to regulate mitochondrial pore formation and cytochrome c release. Here we used a wide spectrum of nicotinic receptor subunit-specific antibodies to show that mitochondria express several nicotinic receptor subtypes in a tissue-specific manner: brain and liver mitochondria contain α7β2, α4β2 and less α3β2 nicotinic receptors, while mitochondria from the lung express preferentially α3β4 receptor subtype; all of them are non-covalently connected to voltage-dependent anion channels and control cytochrome c release. By using selective ligands of different nicotinic receptor subtypes (acetylcholine (1 μM) or dihydro-β-erythroidine (1 μM) for α4β2), conotoxin MII (1 nM) for α3β2, MLA (50 nM) for α7β2 and acetylcholine (10 μM) for all subtypes) and apoptogenic agents triggering different mitochondrial signaling pathways (1 μM wortmannin, 90 μM Ca2+ or 0.5 mM H2O2) it was found that α7β2 receptors affect mainly PI3K/Akt pathway, while α3β2 and α4β2 nAChRs also significantly influence CaKMII- and Src-dependent pathways. It is concluded that cholinergic regulation in mitochondria is realized through multiple nicotinic receptor subtypes, which control various pathways inducing mitochondrial type of apoptosis. © 2014 Elsevier Ltd.

Agency: European Commission | 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.

Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: NMP-2008-1.3-2 | Award Amount: 3.10M | Year: 2009

The purpose of this project is to identify and rate important forms of nanotechnology-related environmental pollution and health hazards that could result from activities involved in nano-structures throughout their life-cycle, and to suggest means that might reduce or eliminate these impacts. Besides the positive multipurpose nano-reinforcement in materials and expanded devices applications, little is known about the environmental and health risks of certain manufactured nanomaterials. Initial research has indicated that nanomaterials can have a negative impact on human health and environmental pollution. For instance, carbon nanotubes may be more toxic than other carbon particles or quartz dust when being absorbed into the lung tissue; however, specific detailed research is required. More importantly, and fundamental to the success of nanotechnology, is the perceived safety of the technology by the public. As activity shifts from research to the development of applications, there exists an urgent need to understanding and managing the associated risks, but in particular to personnel working with these materials. To address these issues, an investigation of biological interactions of nanoscale and nanostructured materials on in vitro toxicological mechanisms is proposed. Further, an assessment of their impact on environmental pollution regarding water, soil and air is also proposed.

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.

Dekaliuk M.O.,Palladin Institute of Biochemistry | Viagin O.,Institute for Scintillation Materials of Ukraine | Malyukin Y.V.,Institute for Scintillation Materials of Ukraine | Demchenko A.P.,Palladin Institute of Biochemistry
Physical Chemistry Chemical Physics | Year: 2014

Despite many efforts, the mechanisms of light absorption and emission of small fluorescent carbon nanoparticles (C-dots) are still unresolved and are a subject of active discussion. In this work we address the question as to whether the fluorescence is a collective property of these nanoparticles or they are composed of assembled individual emitters. Selecting three types of C-dots with "violet", "blue" and "green" emissions and performing a detailed study of fluorescence intensity, lifetime and time-resolved anisotropy as a function of excitation and emission wavelengths together with the effect of viscogen and dynamic fluorescence quencher, we demonstrate that the C-dots represent assemblies of surface-exposed fluorophores. They behave as individual emitters, display electronic anisotropy, do not exchange their excited-state energies via homo-FRET and possibly display sub-nanosecond intra-particle mobility. This journal is © the Partner Organisations 2014.

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.

Gergalova G.,Palladin Institute of Biochemistry | Lykhmus O.,Palladin Institute of Biochemistry | Komisarenko S.,Palladin Institute of Biochemistry | Skok M.,Palladin Institute of Biochemistry
International Journal of Biochemistry and Cell Biology | Year: 2014

Nicotinic acetylcholine receptors are ligand-gated ion channels found in the plasma membrane of both excitable and non-excitable cells. Previously we reported that nicotinic receptors containing α7 subunits were present in the outer membranes of mitochondria to regulate the early apoptotic events like cytochrome c release. Here we show that signaling of mitochondrial α7 nicotinic receptors affects intramitochondrial protein kinases. Agonist of α7 nicotinic receptors PNU 282987 (30 nM) prevented the effect of phosphatidyl inositol-3-kinase inhibitor wortmannin, which stimulated cytochrome c release in isolated mouse liver mitochondria, and restored the Akt (Ser 473) phosphorylation state decreased by either 90 μM Ca2+ or wortmannin. The effect of PNU 282987 was similar to inhibition of calcium-calmodulin-dependent kinase II (upon 90 μM Ca2+) or of Src kinase(s) (upon 0.5 mM H2O2) and of protein kinase C. Cytochrome c release from mitochondria could be also attenuated by α7 nicotinic receptor antagonist methyllicaconitine or α7-specific antibodies. Allosteric modulator PNU 120526 (1 μM) did not improve the effect of agonist PNU 282987. Acetylcholine (1 μM) and methyllicaconitine (10 nM) inhibited superoxide release from mitochondria measured according to alkalization of Ca2+-containing medium. It is concluded that α7 nicotinic receptors regulate mitochondrial permeability transition pore formation through ion-independent mechanism involving activation of intramitochondrial PI3K/Akt pathway and inhibition of calcium-calmodulin-dependent or Src-kinase-dependent signaling pathways. © 2014 Elsevier Ltd.

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.

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.

Kalashnyk O.M.,Palladin Institute of Biochemistry | Gergalova G.L.,Palladin Institute of Biochemistry | Komisarenko S.V.,Palladin Institute of Biochemistry | Skok M.V.,Palladin Institute of Biochemistry
Life Sciences | Year: 2012

Aims: Previously we demonstrated that mouse liver mitochondria express functional α7 nicotinic acetylcholine receptors (nAChRs). The aim of this study was to investigate whether the nAChRs are found in mitochondria of non-neuronal human cell lines. Main methods: Three cell lines: U373 (astrocytes), U937 (monocytes) and Daudi (B lymphocytes) were examined by flow cytometry, Cell ELISA and fluorescent confocal microscopy using the antibodies against extracellular epitopes of α3, α4, α7, α9, β2 and β4 nAChR subunits. Key findings: It is shown that the studied cells expressed different sets of nAChR subunits on the plasma membrane suggesting the presence of α7 nAChRs on all cells, of α3β4 nAChRs on U373 cells and of α4β2/α4β4 nAChRs on U937 cells. In addition to nAChRs exposed on the surface, all cells contained a considerable intracellular pool of α3- and α7-containing nAChRs. The binding of α3-, α7- and β4-specific antibodies partially overlapped with that of mitochondrial outer membrane translocase-specific antibody. Binding of α7-specific antibody also overlapped with that of MitoTracker Green, which binds to active mitochondria. Significance: The data obtained suggest that a part of intracellular α3β4 and α7 nAChRs in U373, U937 and Daudi cells is located on mitochondria. This finding complements our previous observation of α7 nAChRs in mouse liver mitochondria and reveals new intracellular targets for cholinergic regulation. © 2012 Elsevier Inc.

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