Publications by authors named "Vladimir I Popenko"

18 Publications

  • Page 1 of 1

Pre-Senescence Induction in Hepatoma Cells Favors Hepatitis C Virus Replication and Can Be Used in Exploring Antiviral Potential of Histone Deacetylase Inhibitors.

Int J Mol Sci 2021 Apr 27;22(9). Epub 2021 Apr 27.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions-from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.
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http://dx.doi.org/10.3390/ijms22094559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123837PMC
April 2021

Interaction of 5-substituted pyrimidine nucleoside analogues and M.Tuberculosis: A view through an electron microscope.

Biochimie 2020 Apr - May;171-172:170-177. Epub 2020 Mar 6.

Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, 32 Vavilov St., Moscow, 119991, Russia. Electronic address:

The data of transmission electron microscopy (TEM) on morphology of M. tuberculosis H37Rv bacterial cells treated with four analogues of pyrimidine nucleosides with different substituents at 5 position of base are presented. We showed that the growth of M. tuberculosis H37Rv cells effectively inhibited by each of these compounds. This process is accompanied with the accumulation of lipid intracellular vacuole-like inclusions in the cells, appearance of deep protrusions and indentations on the surface, partial and/or complete destruction of the three-layered cell envelope. The exact molecular mechanism of action of 5-substituted pyrimidine nucleosides on M. tuberculosis cells remains to be proved. However, one can suggest that mechanism of action for these compounds is related either to their direct interactions with bacteria cell walls or to interactions with enzymes participating in the process of cell wall formation.
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http://dx.doi.org/10.1016/j.biochi.2020.03.004DOI Listing
December 2020

L-Lysine-modified FeO nanoparticles for magnetic cell labeling.

Colloids Surf B Biointerfaces 2020 Jun 19;190:110879. Epub 2020 Feb 19.

Postovsky Institute of Organic Synthesis, Russian Academy of Sciences (Ural Branch), 22 S. Kovalevskoy St., Yekaterinburg, 620990, Russia; Institute of Chemical Engineering, Ural Federal University, 19 Mira St., Yekaterinburg, 620002, Russia.

The efficiency of magnetic labeling with L-Lys-modified FeO magnetic nanoparticles (MNPs) and the stability of magnetization of rat adipose-derived mesenchymal stem cells, lineage-negative (Lin(-)) hematopoietic progenitor cells from mouse bone marrow and human leukemia K562 cells were studied. For this purpose, covalent modification of MNPs with 3-aminopropylsilane and N-di-Fmoc-L-lysine followed by removal of N-protecting groups was carried out. Since the degree of hydroxylation of the surface of the starting nanoparticles plays a crucial role in the silanization reaction and the possibility of obtaining stable colloidal solutions. In present work we for the first time performed a comparative qualitative and quantitative evaluation of the number of adsorbed water molecules and hydroxyl groups on the surface of chemically and physically obtained FeO MNPs using comprehensive FTIR spectroscopy and thermogravimetric analysis. The results obtained can be further used for magnetic labeling of cells in experiments in vitro and in vivo.
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http://dx.doi.org/10.1016/j.colsurfb.2020.110879DOI Listing
June 2020

Two Receptors, Two Isoforms, Two Cancers: Comprehensive Analysis of KIT and TrkA Expression in Neuroblastoma and Acute Myeloid Leukemia.

Front Oncol 2019 18;9:1046. Epub 2019 Oct 18.

Department of Cancer Cell Biology, Engelhardt Institute of Molecular Biology, RAS, Moscow, Russia.

Pediatric cancers represent a wide variety of different tumors, though they have unique features that distinguish them from adult cancers. Receptor tyrosine kinases KIT and TrkA functions in AML and NB, respectively, are well-characterized. Though expression of these receptors is found in both tumors, little is known about KIT function in NB and TrkA in AML. By combining gene enrichment analysis with multidimensional scaling we showed that pediatric AMLs with t(8;21) or inv16 and high expression levels stand out from other AML subtypes as they share prominent transcriptomic features exclusively with KIT-overexpressing NBs. We showed that AML cell lines had a predominant expression of an alternative TrkAIII isoform, which reportedly has oncogenic features, while NB cell lines had dominating TrkAI-II isoforms. NB cells, on the other hand, had an abnormal ratio of KIT isoforms as opposed to AML cells. Both SCF and NGF exerted protective action against doxorubicin and cytarabine for t(8;21) AML and NB cells. We identified several gene sets both unique and common for pediatric AML and NB, and this expression is associated with KIT or TrkA levels. , and genes are differentially expressed in NBs with high KIT expression and are associated with poor survival in NB. We identified , and genes that are connected with TrkA expression and are marker genes of poor outcome in AML. We also report that , and gene expression is associated with TrkA or KIT expression levels in both AML and NB, and these genes have a prognostic value for both cancers. Thus, we have provided a comprehensive characterization of TrkA and KIT expression along with the oncogenic signatures of these genes across two pediatric tumors.
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http://dx.doi.org/10.3389/fonc.2019.01046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813278PMC
October 2019

Novel 5'-Norcarbocyclic Pyrimidine Derivatives as Antibacterial Agents.

Molecules 2018 Nov 23;23(12). Epub 2018 Nov 23.

Department of Chemistry & Biochemistry, University of Maryland, 1000 Hilltop Circle, Baltimore, MD 21250, USA.

A series of novel 5'-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of was completely inhibited by the most active compounds at a MIC of 67 μg/mL (mc²155) and a MIC of 6.7⁻67 μg/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of ATCC 25177 (MIC 28⁻61 μg/mL) and ATCC 35737 (MIC 50⁻60 μg/mL), as well as two virulent strains of ; a laboratory strain H37Rv (MIC 20⁻50 μg/mL) and a clinical strain with multiple drug resistance MS-115 (MIC 20⁻50 μg/mL). Transmission electron microscopy (TEM) evaluation of H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.
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http://dx.doi.org/10.3390/molecules23123069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321083PMC
November 2018

Intracellular Localization of Densovirus (BgDV1) Capsid Proteins.

Viruses 2018 07 14;10(7). Epub 2018 Jul 14.

Vavilov Institute of General Genetics Russian Academy of Sciences, 119991 Moscow, Russia.

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.
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http://dx.doi.org/10.3390/v10070370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071259PMC
July 2018

Studies of cytokinin receptor-phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum.

Funct Plant Biol 2018 Jan;45(2):192-202

Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya str. 35, 127276 Moscow, Russia.

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor-phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor-phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.
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http://dx.doi.org/10.1071/FP16292DOI Listing
January 2018

The size of DNA molecules and chromatin organization in the macronucleus of the ciliate Didinium nasutum (Ciliophora).

J Eukaryot Microbiol 2015 Mar-Apr;62(2):260-4. Epub 2014 Sep 8.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.

Pulsed-field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short "gene-sized" DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80-265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.
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http://dx.doi.org/10.1111/jeu.12161DOI Listing
June 2015

Lipophilic derivatives of natural chlorins: synthesis, mixed micelles with phospholipids, and uptake by cultured cells.

Bioorg Med Chem 2013 Sep 14;21(17):5420-7. Epub 2013 Jun 14.

Orekhovich Institute of Biomedical Chemistry RAMS, 10, Pogodinskaya Street, 119121 Moscow, Russia.

The chemical synthesis of six lipophilic conjugates of chlorins was carried out, in which lipophilic fragment (either hexadecyl- or cholest-5-en-3β-yloxyethyl-) bound to 13(1)-, 15(2)-, 17(3)-positions of macrocycle by formation of related carboxamides. Structure of synthesized conjugates was studied by spectral methods and molecular modeling. Lipophilic conjugates of chlorins, being mixed with egg yolk phosphatidyl choline, formed mixed micelles stable in aqueous media under physiological conditions. Mixed micelles of conjugates with phosphatidyl choline differing in stoichiometric compositions were prepared and characterized by absorption spectra, electron microscopy and laser scattering. These micelles were found to bind and internalized by human breast carcinoma MCF-7 cells. The presented data reveal that modification of macrocycle with lipophilic substituents, solubilization of obtained conjugates in aqueous medium as mixed micelles with phospholipids, and transfer of mixed micelles to cells is simple approach for targeting of chlorin derivatives, which apparently may be used in photodynamic therapy.
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http://dx.doi.org/10.1016/j.bmc.2013.06.016DOI Listing
September 2013

Anna S. Tikhonenko: Electron microscopist extraordinary.

Bacteriophage 2013 Jan;3(1):e23646

Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.

Anna Sergeyevna Tikhonenko (1925-2010) is to be remembered for the excellency of her electron microscopical work, particularly with bacteriophages. She published 113 articles and one book, (Izdadelstvo Nauka, Moscow 1968; Plenum Press, New York, 1972). It included 134 micrographs and a complete overview of the 316 phages then examined by electron microscopy. Most micrographs were of exceptional quality. This book, a rarity in those days of strict separation of Soviet and Western research, was the first bacteriophage atlas in the literature and presented a morphological classification of phages into five categories of family level, similar to a scheme presented in 1965 by D.E. Bradley (J Royal Microsc Soc 84:257-316). Her book remains one of the fundamentals of phage research.
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http://dx.doi.org/10.4161/bact.23646DOI Listing
January 2013

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora).

Biol Res 2013 ;46(1):69-74

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia.

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.
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http://dx.doi.org/10.4067/S0716-97602013000100010DOI Listing
October 2014

Application of fusion protein 4D5 scFv-dibarnase:barstar-gold complex for studying P185HER2 receptor distribution in human cancer cells.

Biochimie 2012 Aug 17;94(8):1833-6. Epub 2012 Apr 17.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str.32, 119991 Moscow, Russia.

Overexpression of the P185(HER2) protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185(HER2) in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185(HER2) domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au. The distribution of P185(HER2) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was studied at 4 °C and 37 °C. It was shown that at 4 °C the protein P185(HER2) occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185(HER2) caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185(HER2) was located in the coated pits and vesicles, endosomes and multivesicular bodies. The data obtained indicate that the supramolecular 4D5 scFv-dibarnase:barstar-gold complex can be used as a new immunodetection system for exploring the P185(HER2) distribution.
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http://dx.doi.org/10.1016/j.biochi.2012.04.011DOI Listing
August 2012

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin.

Biochimie 2012 May 11;94(5):1180-8. Epub 2012 Feb 11.

A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninsky Gory, Moscow, 119992, Russia.

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A (116)KSKRKKKNKK(125) and B (175)KKATKKESKKQTK(187) reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein-protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.
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http://dx.doi.org/10.1016/j.biochi.2012.02.005DOI Listing
May 2012

The Role of Microtubule Association in Plasmodesmal Targeting of Potato mop-top virus Movement Protein TGBp1.

Open Virol J 2011 5;5:1-11. Epub 2011 Jan 5.

Department of Virology, Biological Faculty, Moscow State University, Moscow, 119992, Russia.

Cell-to-cell movement of Potato mop-top virus (PMTV) is mediated by three virus-encoded 'triple gene block' (TGB) proteins termed TGBp1, TGBp2 and TGBp3. TGBp1 binds virus RNAs to form viral ribonucleoprotein complexes (vRNPs), the transport form of viral genome. TGBp2 and TGBp3 are necessary for intracellular delivery of TGBp1-containing vRNPs to plasmodesmata. To analyze subcellular localization and transport of TGBp1 we used a single binary vector for agrobacterium-mediated co-expression of PMTV TGBp1 fused to green fluorescent protein and TGBp2/TGBp3. At two days post infiltration (dpi) TGBp1 was found in the nucleus and in association with microtubules (MTs). Similar localization pattern was revealed in cells expressing GFP-TGBp1 alone after particle bombardment. At 3 dpi, in addition to the nucleus and MTs, TGBp1 was detected in numerous granular bodies located both along the MTs and at the cell wall. The latter structures co-localized with plasmodesmata-associated callose depositions. At 4 dpi, GFP-TGBp1 was detected in cell wall-associated bodies and also in residual MTs, the nucleoplasm and large perinuclear inclusions resembling aggresomes. Therefore GFP-TGBp1 association with MTs preceded to its localization to plasmodesmata. Disassembly of cell MTs by colchicine prevented GFP-TGBp1 targeting to plasmodesmata and the MT-dependent aggresome formation. Deletion analysis also revealed a correlation between TGBp1 microtubule association and plasmodesmata targeting. We propose that TGBp1 interaction with MTs may be important for the formation of vRNP bodies destined for the transport to plasmodesmata as well as degradation of the excessive TGBp1.
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http://dx.doi.org/10.2174/1874357901105010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109696PMC
July 2011

Barnase as a new therapeutic agent triggering apoptosis in human cancer cells.

PLoS One 2008 Jun 18;3(6):e2434. Epub 2008 Jun 18.

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

Background: RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.

Methodology/principal Findings: In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.

Conclusions/significance: These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002434PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2413406PMC
June 2008

Nucleolar apparatus in the macronucleus of Didinium nasutum (Ciliata): EM and 3D reconstruction.

Protist 2006 Oct 14;157(4):391-400. Epub 2006 Aug 14.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, 119991 Moscow, Russia.

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.
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http://dx.doi.org/10.1016/j.protis.2006.06.001DOI Listing
October 2006

Spontaneous DNA-DNA interaction of homologous duplexes and factors affecting the result of heteroduplex formation.

Biol Chem 2006 Mar;387(3):257-61

Institute of Carcinogenesis, Blokhin Cancer Research Centre, Russian Academy of Medical Sciences, Kashirskoye Shosse 24, Moscow 115478, Russia.

Mutation detection and mismatch repair investigations based on heteroduplex formation require a linear DNA structure. DNA branching, described previously under physiological conditions, has been analysed in the heteroduplex formation process. Symmetrical chi-structures were detected after heteroduplex formation by gel electrophoresis and electron microscopy. Buffer composition, DNA concentration and duplex end-sequences influence DNA branching. Duplexes with homologous central regions but non-complementary ends do not form hybrid heteroduplexes or hybrid Holliday junctions. Our results explain the requirements for efficient heteroduplex formation, which were previously determined empirically: special solution composition, optimal DNA concentration and GC clamps. This provides the theoretical background for further optimisation of the procedure.
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http://dx.doi.org/10.1515/BC.2006.034DOI Listing
March 2006

Mechanism of spontaneous DNA-DNA interaction of homologous linear duplexes.

Biochemistry 2002 Jun;41(24):7795-801

Institute of Carcinogenesis, Blokhin Cancer Research Center RAMS, Kashirskoye shosse 24, Moscow 115478, Russia.

Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.
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http://dx.doi.org/10.1021/bi015959tDOI Listing
June 2002
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