Феофанов Алексей Валерьевич

Личная информация

Работает в ИБХ РАН с 1991 г., является автором более 100 научных публикаций и 12 патентов. А. В. Феофанов преподает курсы "Введение в методы микроскопии в биологии. Оптическая микроскопия ",   «Микроскопия и микроспектроскопия биообъектов. Детекция наночастиц.» студентам Биологического факультета МГУ им. М. В. Ломоносова. 

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
1982–1988 Россия, Москва Московский инженерно-физический институт (МИФИ) диплом инженера-физика
1991 Россия, Москва Московский государственный университет имени М.В. Ломоносова (МГУ), биологический факультет Присуждена учёная степень кандидата физ.-мат. наук
2006 Россия, Москва Московский государственный университет имени М.В. Ломоносова (МГУ), биологический факультет Присуждена учёная степень доктора биологических наук

Научные интересы

Изучение структуры и функциональных свойств биологически активных соединений с широким использованием методов оптической спектроскопии и микроскопии. Разработка новых методических подходов к изучению биологических молекул на основе методов оптической микроскопии и спектроскопии.

 

Основные направления исследований включают в себя:

скрининг, структурно-функциональные, доклинические и клинические исследования новых фотосенсибилизаторов для противоопухолевой и антимикробной фотодинамической терапии (ФДТ); 

оптимизация структуры и изучение свойств конъюгатов хлорина е6 с наночастицами бора применительно к ФДТ, бор нейтрон-захватной терапии рака и флуоресцентной диагностике;

изучение функциональной роли трансмембранных доменов эфриновых тирозинкиназных рецепторов EphA2; 

изучение свойств и механизмов действия на клетки эукариот и прокариот природных пептидов из ядов насекомых;

поиск новых лигандов потенциал-зависимых калиевых каналов, изучение их активности и свойств.   

Основные научные результаты

В области оптической спектроскопии А. В. Феофановым выполнена серия работ, посвященных разработке методик резонансного комбинационного рассеяния (КР) с возбуждением в УФ области для исследования биологических молекул, методик создания ГКР-активных субстратов с повышенной устойчивостью в органических средах, которые дали возможность применять спектроскопию гигантского комбинационного рассеяния (ГКР) для изучения молекулярных систем в органических растворителях.

В работах А. В. Феофанова предложены и успешно апробированы оригинальные методики, позволяющие по спектрам флуоресценции изучать молекулярные взаимодействия биологически-активных соединений (БАС) в живых клетках с субмикронным пространственным разрешением. Разработаны уникальные методы количественной оценки внутриклеточной концентрации БАС и их комплексов на основе анализа спектров с привлечением данных моделирования молекулярных взаимодействий в растворах. На основе проведенных исследований сформированы универсальные принципы нового направления оптической микроскопии, обеспечивающего неразрушающий количественный анализ БАС и их молекулярных взаимодействий в живых клетках.

Под руководством А. В. Феофанова разработана и широко применяется схема комплексного сравнительного исследования молекулярных и клеточных свойств фотосенсибилизаторов (ФС), позволяющая выяснить влияние структуры молекулы на способность образовывать комплексы с белками, нуклеиновыми кислотами и липидными структурами, на способность продуцировать в составе этих комплексов активные формы кислорода, проникать в раковые клетки и накапливаться там в фотодинамически-активной форме, а также позволяющая изучать взаимосвязи между внутриклеточной локализацией ФС и механизмами фотоиндуцированной гибели клеток.

Избранные публикации

  1. AllardVannier E., HervéAubert K., Kaaki K., Blondy T., Shebanova A., Shaitan K.V., Ignatova A.A., Saboungi M.L., Feofanov A.V., Chourpa I. (2016). Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis. Biochim. Biophys. Acta , [+]

    This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA.

    ID:1689
  2. Nekrasova O., Kudryashova K., Fradkov A., Yakimov S., Savelieva M., Kirpichnikov M., Feofanov A. (2016). Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels. J. Biotechnol. 241, 127–135 [+]

    Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

    ID:1690
  3. Valieva M.E., Armeev G.A., Kudryashova K.S., Gerasimova N.S., Shaytan A.K., Kulaeva O.I., McCullough L.L., Formosa T., Georgiev P.G., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. (2016). Large-scale ATP-independent nucleosome unfolding by a histone chaperone. Nat. Struct. Mol. Biol. , [+]

    DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy. FACT binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

    ID:1625
  4. Shulepko M.A., Lyukmanova E.N., Shenkarev Z.O., Dubovskii P.V., Astapova M.V., Feofanov A.V., Arseniev A.S., Utkin Y.N., Kirpichnikov M.P., Dolgikh D.A. (2016). Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana. Protein Expr. Purif. 130, 13–20 [+]

    Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of (13)C,(15)N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

    ID:1599
  5. Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Y.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. (2016). Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol , [+]

    Разработан комплексный подход к поиску, исследованию и конструированию  пептидных блокаторов калиевого канала Kv1.6. Подход основан на применении разработанной нами биоинженерной аналитической системы для изучения связывания блокаторов с гибридным каналом KcsA-Kv1.6 методом конфокальной микроскопии и молекулярного моделирования комплексов пептидных блокаторов с каналом Kv1.6. Используя разработанный подход, охарактеризована аффинность ряда пептидных блокаторов к каналу Kv1.6, построены молекулярные модели их комплексов, описан интерфейс взаимодействия и аминокислотные остатки, влияющие на селективность  взаимодействия блокаторов с каналом Kv1.6.

    ID:1611
  6. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

    ID:1420
  7. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. (2016). Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels. Sci Rep 6, 33314 [+]

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

    ID:1561
  8. Dubovskii P.V., Vassilevski A.A., Kozlov S.A., Feofanov A.V., Grishin E.V., Efremov R.G. (2015). Latarcins: versatile spider venom peptides. Cell. Mol. Life Sci. 72 (23), 4501–22 [+]

    Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined.

    ID:1395
  9. Kuzmenkov A.I., Vassilevski A.A., Kudryashova K.S., Nekrasova O.V., Peigneur S., Tytgat J., Feofanov A.V., Kirpichnikov M.P., Grishin E.V. (2015). Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY. J. Biol. Chem. 290 (19), 12195–209 [+]

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs.

    ID:1310
  10. Kudryashova K.S., Chertkov O.V., Nikitin D.V., Pestov N.A., Kulaeva O.I., Efremenko A.V., Solonin A.S., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. (2015). Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET. Methods Mol. Biol. 1288, 395–412 [+]

    Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.

    ID:1471
  11. Pluzhnikov K.A., Kozlov S.A., Vassilevski A.A., Vorontsova O.V., Feofanov A.V., Grishin E.V. (2014). Linear antimicrobial peptides from Ectatomma quadridens ant venom. Biochimie 107 Pt B, 211–5 [+]

    Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of ∼105 μg/g.

    ID:1150
  12. Кузнецов А.С., Дубовский П.В., Воронцова О.В., Феофанов А.В., Ефремов Р.Г. (2014). Взаимодействие линейных катионных пептидов с фосфолипидными мембранами и полимерами сиаловой кислоты. Биохимия 79 (5), 583–594583–594 ID:1123
  13. Sharonov G.V., Bocharov E.V., Kolosov P.M., Astapova M.V., Arseniev A.S., Feofanov A.V. (2014). Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase. J. Biol. Chem. 289 (21), 14955–64 [+]

    The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.

    ID:1139
  14. Hoang A.N., Vo H.D., Vo N.P., Kudryashova K.S., Nekrasova O.V., Feofanov A.V., Kirpichnikov M.P., Andreeva T.V., Serebryakova M.V., Tsetlin V.I., Utkin Y.N. (2014). Vietnamese Heterometrus laoticus scorpion venom: evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel. Toxicon 77, 40–8 [+]

    The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K⁺-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (K(i) 59 nM) to Kv1.3 potassium channel.

    ID:1082
  15. Efremenko A.V., Ignatova A.A., Grin M.A., Sivaev I.B., Mironov A.F., Bregadze V.I., Feofanov A.V. (2014). Chlorin e6 fused with a cobalt-bis(dicarbollide) nanoparticle provides efficient boron delivery and photoinduced cytotoxicity in cancer cells. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology 13 (1), 92–102 [+]

    Further development of boron neutron capture therapy (BNCT) requires new neutronsensitizers with improved ability to deliver (10)B isotopes in cancer cells. Conjugation of boron nanoparticles with porphyrin derivatives is an attractive and recognized strategy to solve this task. We report on breakthroughs in the structural optimization of conjugates of chlorin e6 derivative with cobalt-bis(dicarbollide) nanoparticles resulting in the creation of dimethyl ester 13-carbomoylchlorin e6 [N-hexylamine-N'-ethoxyethoxy]-cobalt-bis(dicarbollide) (conjugate 1). Conjugate 1 is able to accumulate quickly and efficiently (distribution factor of 80) in cancer cells, thus delivering more than 10(9) boron atoms per cell when its extracellular concentration is more than 1 μmol L(-1). Also 1 is an active photosensitizer and is phototoxic towards human lung adenocarcinoma A549 cells at 80 nmol L(-1) (50% cell death). Photoinduced cytotoxicity of 1 is associated with lipid peroxidation, lysosome rupture and protease activity enhancement. Conjugate 1 fluoresces in the red region (670 nm), which is useful to monitor its accumulation and distribution in vivo. It is not toxic to cells without activation by neutrons or photons. Structural features that improve the functional properties of 1 are discussed. The properties of 1 warrant its preclinical evaluation as a multifunctional agent for BNCT, photodynamic therapy and fluorescent tumor diagnosis.

    ID:1692
  16. Vassilevski A.A., Sachkova M.Y., Ignatova A.A., Kozlov S.A., Feofanov A.V., Grishin E.V. (2013). Spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of molecules identified in the lynx spider Oxyopes takobius. FEBS J. 280 (23), 6247–61 [+]

    In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 μg·g(-1) ) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 μm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins.

    ID:980
  17. Kudryashova K.S., Nekrasova O.V., Kuzmenkov A.I., Vassilevski A.A., Ignatova A.A., Korolkova Y.V., Grishin E.V., Kirpichnikov M.P., Feofanov A.V. (2013). Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study. Analytical and bioanalytical chemistry , [+]

    Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

    ID:782
  18. Nekrasova O.V., Sharonov G.V., Tikhonov R.V., Kolosov P.M., Astapova M.V., Yakimov S.A., Tagvey A.I., Korchagina A.A., Bocharova O.V., Wulfson A.N., Feofanov A.V., Kirpichnikov M.P. (2012). Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity. Biochemistry Mosc. 77 (12), 1387–94 [+]

    Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

    ID:1479
  19. Efremenko A.V., Ignatova A.A., Borsheva A.A., Grin M.A., Bregadze V.I., Sivaev I.B., Mironov A.F., Feofanov A.V. (2012). Cobalt bis(dicarbollide) versus closo-dodecaborate in boronated chlorin e(6) conjugates: implications for photodynamic and boron-neutron capture therapy. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology 11 (4), 645–652 [+]

    Conjugation of boron nanoparticles with porphyrins is an attractive way to create dual agents for anticancer boron neutron capture therapy (BNCT) and photodynamic therapy (PDT). Properties of chlorin e(6) conjugated with two cobalt bis(dicarbollide) nanoparticles (1) or with a closo-dodecaborate nanoparticle (2) are reported. Fluorescent dianionic conjugates 1 and 2 penetrate in A549 human lung adenocarcinoma cells, stain cytoplasm diffusely and accumulate highly in lysosomes but are not toxic themselves for cells. Average cytoplasmic concentration of boron atoms (B) achieves 270 μM (ca. 2 × 10(8) B/cell) and 27 μM (ca. 2 × 10(7) B/cell) at the 1.5 μM extracellular concentration of 1 and 2, respectively, that makes conjugate 1 especially suitable for BNCT. Conjugate 2 causes photoinduced cell death at micromolar concentrations and can be considered also as a photosensitizer for PDT. Conjugates 1 and 2 have high quantum yields of singlet oxygen generation (0.55 and 0.85 in solution, respectively), identical intracellular localization and similar lipid-like microenvironment but conjugate 1 possesses no photoinduced cytotoxicity. A presence of cobalt complexes in conjugate 1 is supposed to be a reason of the observed antioxidative effect in cellular environment, but an exact mechanism of this intriguing phenomenon is unclear. Due to increased intracellular accumulation and absence of photoinduced cytotoxicity conjugate 1 is promising for fluorescence diagnostics of cancer.

    ID:783
  20. Dubovskii P.V., Vassilevski A.A., Samsonova O.V., Egorova N.S., Kozlov S.A., Feofanov A.V., Arseniev A.S., Grishin E.V. (2011). Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin. FEBS J. 278 (22), 4382–93 [+]

    A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs. DATABASE: The protein sequence of oxyopinin 4a (Oxt 4a) has been submitted to the UniProt Knowledgebase (UniProtKB) under the accession number P86350. The coordinates and chemical shifts of Oxt 4a in complex with dodecylphosphocholine micelles have been deposited in the Protein Data Bank and Biological Magnetic Resonance Bank under the accession codes 2L3I and 17194, respectively. The nucleotide sequence encoding Oxt 4a has been submitted to the EMBL Nucleotide Sequence Database under the accession number FN997582.

    ID:546
  21. Nolde S.B., Vassilevski A.A., Rogozhin E.A., Barinov N.A., Balashova T.A., Samsonova O.V., Baranov Y.V., Feofanov A.V., Egorov T.A., Arseniev A.S., Grishin E.V. (2011). Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli). J. Biol. Chem. 286 (28), 25145–53 [+]

    This study presents purification, activity characterization, and (1)H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.

    ID:543
  22. Vorontsova O.V., Egorova N.S., Arseniev A.S., Feofanov A.V. (2011). Haemolytic and cytotoxic action of latarcin Ltc2a. Biochimie 93 (2), 227–241 [+]

    Activity and action mechanisms of latarcin 2a (Ltc2a), an antimicrobial peptide from the venom of the spider Lachesana tarabaevi (Zodariidae), were studied in vitro on human cells. Cytotoxicity of Ltc2a for erythrocytes (EC(50) = 3.4 μM), leukocytes (EC(50) = 19.5 μM) and erythroleukemia K562 cells (EC(50) = 3.3 μM) has been found to be primary related to plasma membrane destabilization. Using fluorescently labeled Ltc2a, three common features are found for erythrocytes and K562 cells: pronounced inhomogeneity of cellular response to Ltc2a; complex multistage character of Ltc2a-cell interactions; a positive feedback between Ltc2a binding to plasma membrane and development of toxic effects. Discocyte - echinocyte - spherocyte - ghost is a sequence of Ltc2a-induced transformations of erythrocytes that are accompanied by multistage enhancement of Ltc2a membrane binding, formation of small (ca. 2.0 nm) membrane pores, osmotic imbalance development and reorganization of the pores into large (ca. 13 nm) membrane openings that are preserved in ghosts. Ltc2a induces membrane blebbing and swelling of K562 cells followed by cell death. Cytotoxic action occurs through formation of membrane pores (ca. 3.7 nm) which show greater permeability for anionic than cationic molecules. The pore formation is accompanied with self-assisted Ltc2a internalization and accumulation in mitochondria, mitochondrion inactivation and apoptosis-independent phosphatidylserine externalization.

    ID:784
  23. Vassilevski A.A., Fedorova I.M., Maleeva E.E., Korolkova Y.V., Efimova S.S., Samsonova O.V., Schagina L.V., Feofanov A.V., Magazanik L.G., Grishin E.V. (2010). Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE. J. Biol. Chem. 285 (42), 32293–302 [+]

    Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

    ID:394
  24. Polyansky A.A., Vassilevski A.A., Volynsky P.E., Vorontsova O.V., Samsonova O.V., Egorova N.S., Krylov N.A., Feofanov A.V., Arseniev A.S., Grishin E.V., Efremov R.G. (2009). N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins. FEBS Lett. 583 (14), 2425–8 [+]

    In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

    ID:318
  25. Nekrasova O.V., Ignatova A.A., Nazarova A.I., Feofanov A.V., Korolkova Y.V., Boldyreva E.F., Tagvei A.I., Grishin E.V., Arseniev A.S., Kirpichnikov M.P. (2009). Recombinant Kv channels at the membrane of Escherichia coli bind specifically agitoxin2. J Neuroimmune Pharmacol 4 (1), 83–91 [+]

    Potassium voltage-gated channels (Kv) are considered as molecular targets in a number of serious neuronal, immune, and cardiac disorders. Search for efficient low-molecular weight modulators of Kv channel function provides a basis for the development of an appropriate therapy for various Kv-mediated diseases. We report here on a new bacterial cell-based system, which is suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. To create this system, high-level expression of KcsA-Kv1.3 and KcsA-Kv1.1 hybrid proteins (ligand-binding sites of Kv1.3 or Kv1.1 fused with prokaryotic KcsA potassium channel) was achieved in the plasma membrane of Escherichia coli. An efficient procedure of E. coli conversion to intact spheroplasts was developed. We demonstrate that fluorescently labeled agitoxin 2 binds specifically to high-affinity and lower-affinity sites of KcsA-Kv1.3 and KcsA-Kv1.1, respectively, at the membrane of spheroplasts. Number of binding sites per cell is estimated to be (1.0 +/- 0.6) x 10(5) and (0.3 +/- 0.2) x 10(5) for KcsA-Kv1.3- and KcsA-Kv1.1-presenting cells, respectively, that allows reliable detection of ligand-receptor interactions by confocal laser scanning microscopy. This bacterial cell-based system is intended for screening of ligands to membrane-embedded pharmaceutical targets.

    ID:786
  26. Ignatova A.A., Maslova A.S., Kirpichnikov M.P., Feofanov A.V. (2009). Interaction of the photosensitizer 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) with normal and cancerous blood cells. Bioorg. Khim. 35 (6), 830–836 [+]

    The interaction of 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) (CIC) with normal blood cells and human K562 and HL60 myeloid leukemia cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.

    ID:785
  27. Feofanov A., Charonov S., Fleury F., Kudelina I., Jardillier J.C., Nabiev I. (2009). Confocal spectral imaging analysis of intracellular interactions of mitoxantrone at different phases of the cell cycle. Anticancer Res. 19 (6B), 5341–5348 [+]

    It is suggested that the cytotoxicity of anticancer agent mitoxantrone (MITOX) is related to a complex combination of molecular interactions which lead to slowing of S phase traverse and arresting of cells in G2 phase of the cell cycle or even to an apoptosis at high concentration of MITOX. Here intracellular molecular interactions of MITOX were visualised and studied using the confocal spectral imaging technique in synchronised K562 cells. Localisation, quantitative distributions of MITOX in the polar environment, MITOX bound to hydrophobic cellular structures (MITOXphob), nucleic acid-related complexes of MITOX (MITOXNA) and relative distributions of naphthoquinoxaline (NQX) metabolite and intrinsic cellular fluorescence of porphyrins were measured within cytoplasmic and nuclear compartments (chromosomes) of the G2, S, and M cells treated with 10 or 2 microM of MITOX for 1 hour. Colocalisation of MITOX, NQX metabolite and sites of intrinsic cellular fluorescence indicates an accumulation of MITOX within or near mitochondria. One may suppose that due to high concentration MITOX can compete with natural substrates for binding to the enzymes thus affecting the normal functioning of a mitochondria. A remarkable redistribution of MITOX and its complexes occurs in the M cells. In particular, a prominent amount of MITOX is associated with the surface of chromatids but not with the cytoplasmic structures in M cells. At the present time the exact location of the sites of MITOX accumulation in the M cells is not known. It is thought to be some cytoskeleton/microtubule structures associated directly with the chromosomes. Selective labelling of particular cytoskeleton structures and/or proteins in MITOX treated cells is in the progress now and the question will be addressed using the CSI technique.

    ID:794
  28. Novoseletsky V.N., Volyntseva A.D., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. (2009). Modeling of the Binding of Peptide Blockers to Voltage-Gated Potassium Channels: Approaches and Evidence. Acta Naturae 8 (2), 35–46 [+]

    Modeling of the structure of voltage-gated potassium (KV) channels bound to peptide blockers aims to identify the key amino acid residues dictating affinity and provide insights into the toxin-channel interface. Computational approaches open up possibilities for in silico rational design of selective blockers, new molecular tools to study the cellular distribution and functional roles of potassium channels. It is anticipated that optimized blockers will advance the development of drugs that reduce over activation of potassium channels and attenuate the associated malfunction. Starting with an overview of the recent advances in computational simulation strategies to predict the bound state orientations of peptide pore blockers relative to KV-channels, we go on to review algorithms for the analysis of intermolecular interactions, and then take a look at the results of their application.

    ID:1691
  29. Artemenko E.O., Egorova N.S., Arseniev A.S., Feofanov A.V. (2008). Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment. Biochim. Biophys. Acta 1778 (10), 2361–2367 [+]

    Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide (Kd=7.2+/-0.4 microM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes (DeltaG=-15.4+/-0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.

    ID:787
  30. Vassilevski A.A., Kozlov S.A., Samsonova O.V., Egorova N.S., Karpunin D.V., Pluzhnikov K.A., Feofanov A.V., Grishin E.V. (2008). Cyto-insectotoxins, a novel class of cytolytic and insecticidal peptides from spider venom. Biochem. J. 411 (3), 687–96 [+]

    Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.

    ID:386
  31. Nazarova A., Ignatova A., Feofanov A., Karmakova T., Pljutinskaya A., Mass O., Grin M., Yakubovskaya R., Mironov A., Maurizot J.C. (2007). 13,15-N-cycloimide derivatives of chlorin p6 with isonicotinyl substituent are photosensitizers targeted to lysosomes. Photochem. Photobiol. Sci. 6 (11), 1184–1196 [+]

    Four monocationic cycloimide derivatives of chlorin p(6) (CICD) were studied as photosensitizers and compared to a structurally similar neutral derivative. Cationic CICD are highly photostable (quantum yield of photobleaching is about 1 x 10(-5), generate singlet oxygen under irradiation (quantum yields are 0.3-0.45), can be involved in a photo-induced substrate-dependent generation of superoxide radicals, but do not produce OH . 17,18-delta-lacton 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) () and 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) methyl ester () possess high cancer cell killing photodynamic activity, but they provide no photoinduced bactericidal effect. Substitution of an ethyl group with a hydroxyethyl or acetyl group at position 3 of the macrocycle results in a decrease in extinction and intracellular accumulation that finally leads to the reduced photocytotoxicity. Cationic CICD are targeted to lysosomes, and their intracellular penetration occurs most probably via caveolae-dependent endocytosis. Photodynamic treatment with cationic CICD results in the cell death via necrosis at both sub-phototoxic (40-70% of dead cells) and phototoxic (90-100% of dead cells) regimes of cell treatment. Irradiation induces lysosome damage, leakage of CICD from lysosomes and development of protease activity in cytoplasm, whereas mitochondria are not affected with irradiation. A positive charge of cationic CICD modified drastically an internalization pathway, sites of intracellular localization and mechanisms of photoinduced cytotoxicity as compared to previously studied neutral and anionic CICD. Our experiments with different CICD show that varying charge and structure of substituents it is possible to modulate many cellular properties of CICD in order to find the best molecular template of the advanced near-IR photosensitizer for photodynamic therapy.

    ID:788
  32. Феофанов А.В. (2007). Спектральная лазерная сканирующая конфокальная микроскопия в биологических исследованиях. Успехи биолог. хим. 47, 371–410 [+]

    В работе рассматриваются применения флуоресцентного спектрального анализа в лазерной сканирующей конфокальной микроскопии (ЛСКМ). Спектральная ЛСКМ позволяет идентифицировать и разделять в исследуемых образцах перекрывающиеся сигналы нескольких флуоресцирующих соединений и (или) белков слияния, а также учитывать вклад эндогенной флуоресценции, что существенно повышает чувствительность, надежность и точность анализа по сравнению с традиционной ЛСКМ. Анализ полных спектров флуоресценции дает возможность изучать молекулярные взаимодействия флуоресцирующих биологически активных соединений (БАС) в живых клетках с трехмерным субмикронным пространственным разрешением. Спектральная ЛСКМ позволяет измерять концентрации БАС и их комплексов в живых клетках, оценивать средние концентраций БАС в органоидах, клеточных доменах, в среднем по клетке, а также проводить статистически достоверный анализ клеточных концентраций БАС на ограниченной выборке клеток. Спектральная ЛСКМ является уникальным дополнением к традиционным методам флуоресцентной микроскопии и ЛСКМ.

    ID:91
  33. Kozlov S.A., Vassilevski A.A., Feofanov A.V., Surovoy A.Y., Karpunin D.V., Grishin E.V. (2006). Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J. Biol. Chem. 281 (30), 20983–92 [+]

    Семейство коротких линейных полипептидных молекул — латарцинов — было найдено в яде паука Lachesana tarabaevi, часть из них была получена химическим синтезом и исследована на антибактериальное действие. Бактерицидная активность латарцинов соответствовала лучшим антимикробным пептидам, обнаруженным у различных видов животных. Большой терапевтический потенциал этих простых по структуре полипептидных молекул будет реализован, с большой долей вероятности, в самое ближайшее время.

    ID:86
  34. Sharonov G.V., Karmakova T.A., Kassies R., Pljutinskaya A.D., Grin M.A., Refregiers M., Yakubovskaya R.I., Mironov A.F., Maurizot J.C., Vigny P., Otto C., Feofanov A.V. (2006). Cycloimide bacteriochlorin p derivatives: photodynamic properties and cellular and tissue distribution. Free Radic. Biol. Med. 40 (3), 407–19 [+]

    В работе проведено сравнительное исследование 11 новых фотосенсибилиаторов для фотодинамической терапии рака — циклоимидных производных бактериохлорина р с различными боковыми заместителями. Показано, что данные соединения обладают интенсивным поглощением в спектральном диапазоне прозрачности биологической ткани (780—830 нм), высоким квантовым выходом генерации синглетного кислорода (0,54—0,56) и способностью проникать и накапливаться в опухолевых клетках in vitro. Установлено, что с помощью боковых заместителей можно направлять данные соединения преимущественно в аппарат Гольджи, или липидные капли, или лизосомы и обеспечивать высокие коэффициенты накопления этих соединений в раковых клетках. На модели перевивной опухоли мышей выявлено усиленное накопление изучаемых соединений в опухоли и окружающей соединительной ткани, что с учетом перечисленных выше свойств позволяет рекомендовать циклоимидные производные бактериохлорина р в качестве перспективных фотосенсибилизаторов для фотодинамической терапии рака.

    ID:92
  35. Karmakova T., Feofanov A., Pankratov A., Kazachkina N., Nazarova A., Yakubovskaya R., Lebedeva V., Ruziyev R., Mironov A., Maurizot J.C., Vigny P. (2006). Tissue distribution and in vivo photosensitizing activity of 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester. J. Photochem. Photobiol. B, Biol. 82 (1), 28–36 [+]

    Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.

    ID:789
  36. Feofanov A.V., Sharonov G.V., Astapova M.V., Rodionov D.I., Utkin Y.N., Arseniev A.S. (2005). Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage. Biochem. J. 390 (Pt 1), 11–8 [+]

    Статья посвящена исследованию механизмов цитотоксического действия цитотоксинов (ЦТ) из яда кобр. В данной работе показано, что ЦТ 1 и 2 из яда кобры Naja oxiana, ЦТ 1 из Naja haje и ЦТ 3 из Naja kaouthia способны накапливаться в лизосомах клеток промиелоцитарной лейкемии человека HL60 и аденокарциномы легкого человека A549. Кинетика и концентрационная зависимость накопления ЦТ в лизосомах согласуется с кинетикой и концентрационной зависимостью гибели клеток, что свидетельствует о том, что лизосомы могут быть одной из мишеней ЦТ, а воздействие на эту мишень заключается в концентрационно-зависимой пермеабилизации мембраны лизосом.

    ID:93
  37. Sharonov G.V., Feofanov A.V., Bocharova O.V., Astapova M.V., Dedukhova V.I., Chernyak B.V., Dolgikh D.A., Arseniev A.S., Skulachev V.P., Kirpichnikov M.P. (2005). Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells. Apoptosis 10 (4), 797–808 [+]

    В работе докладывается о разработке метода измерения проапоптотическй активности экзогенного цитохрома с в живых клетках. Метод основан на введении белка в цитоплазму клетки с помощью электропорации и выявлении с помощью флуоресцентной микроскопии признаков развития апоптоза в клетках. С помощью данного метода были измерены относительные про-апоптозные активности лошадиного цитохрома с и четырех мутантных вариантов данного белка. Обнаружено, что аминокислотная замена К72W полностью блокирует про-апоптозную активность цитохрома с, но не влияет на его «дыхательную» функцию. Методом КОМИРСИ была впервые оценена минимальная цитоплазматическая концентрация лошадиного цитохрома с, необходимая для индукции апоптоза в клетках WEHI-3b.

    ID:94
  38. Feofanov A.V., Sharonov G.V., Dubinnyi M.A., Astapova M.V., Kudelina I.A., Dubovskii P.V., Rodionov D.I., Utkin Y.N., Arseniev A.S. (2004). Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje. Biochemistry Mosc. 69 (10), 1148–57 [+]

    Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

    ID:341
  39. Karmakova T., Feofanov A., Nazarova A., Grichine A., Yakubovskaya R., Lukyanets E., Maurizot J.C., Vigny P. (2004). Distribution of metal-free sulfonated phthalocyanine in subcutaneously transplanted murine tumors. J. Photochem. Photobiol. B, Biol. 75 (1-2), 81–87 [+]

    Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H(2)PcS(2.4)) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H(2)PcS(2.4) with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H(2)PcS(2.4) intravenous injection revealed that H(2)PcS(2.4) was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H(2)PcS(2.4) was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H(2)PcS(2.4) less than adjacent skin structures, whereas EC and LLC accumulated H(2)PcS(2.4) alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H(2)PcS(2.4) accumulation. The H(2)PcS(2.4) concentration in remote healthy tissues (skin, muscles and connective tissue) was 2-3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H(2)PcS(2.4) predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.

    ID:790
  40. Feofanov A., Sharonov G., Grichine A., Karmakova T., Pljutinskaya A., Lebedeva V., Ruziyev R., Yakubovskaya R., Mironov A., Refregier M., Maurizot J.C., Vigny P. (2004). Comparative study of photodynamic properties of 13,15-N-cycloimide derivatives of chlorin p6. Photochem. Photobiol. 79 (2), 172–88 [+]

    В работе проведено сравнительное исследование четырех новых фотосенсибилизаторов на основе циклоимидных производных хлорина p6 с различными боковыми заместителями. Установлено, что фотодинамическая активность данных соединений в отношении опухолевых клеток in vitro в несколько сот раз выше, чем активность используемого в клинике препарата Фотогем. Показано, что данные соединения эффективно образуют синглетный кислород (квантовый выход 0,35—0,66) и накапливаются в клетках в мономерной фотоактивной форме в связанном с липидными структурами состоянии. В работе продемонстрировано, что с помощью боковых заместителей можно направлять данные соединения в различные внутриклеточные компартменты: аппарат Гольджи, митохондрии, ядерную мембрану и липидные капли. Под действием света данные фотосенсибилизаторы вызывают гибель клеток преимущественно по механизму апоптоза при сублетальном режиме (гибель 50% клеток) и по механизму некроза — при летальном (гибель 100% клеток).

    ID:95
  41. Chudakov D.M., Feofanov A.V., Mudrik N.N., Lukyanov S., Lukyanov K.A. (2003). Chromophore environment provides clue to "kindling fluorescent protein" riddle. J. Biol. Chem. 278 (9), 7215–9 [+]

    asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

    ID:293
  42. Feofanov A., Grichine A., Karmakova T., Pljutinskaya A., Lebedeva V., Filyasova A., Yakubovskaya R., Mironov A., EgretCharlier M., Vigny P. (2002). Near-infrared photosensitizer based on a cycloimide derivative of chlorin p6: 13,15-N-(3'-hydroxypropyl)cycloimide chlorin p6. Photochem. Photobiol. 75 (6), 633–643 [+]

    The 13,15-N-(3'-hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell-killing activity. It is 43-, 61- and 110-fold more active than chlorin p6, 3-formyl-3-devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002-0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 +/- 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3- to 4-fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 +/- 5 min. The time for 50% release of CIC from the cells is 60 +/- 10 min. A 10-fold decrease in CIC intracellular penetration at 22 degrees C proves that temperature-sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2-1.6 microM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic-type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near-IR photosensitizer.

    ID:791
  43. Feofanov A., Grichine A., Karmakova T., Kazachkina N., Pecherskih E., Yakubovskaya R., Lukyanets E., Derkacheva V., EgretCharlier M., Vigny P. (2002). Chelation with metal is not essential for antitumor photodynamic activity of sulfonated phthalocyanines. Photochem. Photobiol. 75 (5), 527–33 [+]

    It is generally assumed that a central metal is essential for the efficiency of phthalocyanines in photodynamic therapy (PDT) of cancer. Contrary to the set opinion, the results of the present study indicate that the metal-free sulfonated phthalocyanines (H2PcSn, where n is the number of sulfonate groups per molecule) possess a considerable photoactivity. The relative phototoxicities of H2PcS1.5, H2PcS2.4, H2PcS3.1 and H2PcS3.8 on HEp2 human epidermoid carcinoma cells were 3.3, 20, 3.3 and 1, respectively, thus demonstrating dependence of the activity on the sulfonation degree, known for metallo-PcSn. A significant delay in tumor growth and a decrease in tumor regrowth rate were observed in mice after PDT with H2PcS2.4. The antitumor effect declined in the order H2PcS2.4 > H2PcS3.1 > H2PcS1.5 and vanished for H2PcS3.8. We demonstrate here that the high photodynamic activity of H2PcS2.4 can be explained by its physicochemical properties in living cells and tissues. Thus, H2PcSn (n is about 2) can be considered as a new alternative in PDT of light-accessible neoplasms and further clinic-oriented studies are warranted.

    ID:792
  44. Galanina O., Feofanov A., Tuzikov A.B., Rapoport E., Crocker P.R., Grichine A., EgretCharlier M., Vigny P., LePendu J., Bovin N.V. (2001). Fluorescent carbohydrate probes for cell lectins. Spectrochim Acta A Mol Biomol Spectrosc 57 (11), 2285–2296 [+]

    Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe(x)-PAA-flu, Sia2-PAA-flu, GlcNAc2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe(x)-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe(x)-PAA-3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe(x)-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

    ID:793
  45. Feofanov A.V., Grichine A.I., Shitova L.A., Karmakova T.A., Yakubovskaya R.I., EgretCharlier M., Vigny P. (2000). Confocal raman microspectroscopy and imaging study of theraphthal in living cancer cells. Biophys. J. 78 (1), 499–512 [+]

    Binary systems combining a transition metal complex and ascorbate have been proposed recently for catalytic therapy of malignant tumors. The killing effect on tumor cells is achieved by production of free radicals in the course of accelerated oxidation of ascorbate by dioxygen in the presence of transition metal complexes. Further progress in the development of binary catalytic systems (BCSs) requires a special method for their investigation in cells and tissues, because neither component of BCSs fluoresces. Here a resonance Raman confocal spectral imaging (RR CSI) technique was introduced as a unique approach to monitor quantitatively the transition metal complexes within living cells. Intracellular accumulation, localization, and retention of theraphthal (TP), a catalyst of the advanced TP/ascorbate BCS, were investigated in A549 cells with the RR CSI technique. The cellular analysis was complemented with the detailed study of molecular interactions of TP in solution and environmental factors affecting the RR spectrum of TP. TP does not penetrate into membranes, it binds very weakly to DNA and RNA, but it readily forms complexes with proteins. Binding with Ca(2+) cations and decreasing pH below 6 induce aggregation of TP. By analyzing RR spectra recorded from every point within a TP-treated cell, three states of the agent were discriminated, namely, monomeric TP in polar environment, TP bound to proteins, and aggregated TP. Their cytoplasmic and nuclear distributions were mapped at different stages of uptake and efflux. By introducing organelle-selective fluorescent probes into drug-treated cells and measuring intracellular localization of both the probe and the drug, compartmentation of TP was revealed. Cell growth suppression by the TP/ascorbate system was measured, and probable molecular and organelle targets of radical damage were characterized.

    ID:795
  46. Feofanov A., Sharonov S., Fleury F., Kudelina I., Nabiev I. (1997). Quantitative confocal spectral imaging analysis of mitoxantrone within living K562 cells: intracellular accumulation and distribution of monomers, aggregates, naphtoquinoxaline metabolite, and drug-target complexes. Biophys. J. 73 (6), 3328–3336 [+]

    Confocal spectral imaging (CSI) technique was used for quantitative analysis of the uptake, subcellular localization, and characteristics of localized binding and retention of anticancer agent mitoxantrone (MITOX) within human K562 erythroleukemia cells. The CSI technique enables identification of the state and interactions of the drug within the living cells. Utilizing this unique property of the method, intracellular distributions were examined for monomeric MITOX in polar environment, MITOX bound with hydrophobic cellular structures, naphthoquinoxaline metabolite, and nucleic acid-related complexes of MITOX. The features revealed were compared for the cells treated with 2 microM or 10 microM of MITOX for 1 h and correlated to the known data on antitumor action of the drug. MITOX was found to exhibit high tendency to self-aggregation within intracellular media. The aggregates are concluded to be a determinant of long-term intracellular retention of the drug and a source of persistent intracellular binding of MITOX. Considerable penetration of MITOX in the hydrophobic cytoskeleton structures as well as growing accumulation of MITOX bound to nucleic acids within the nucleus were found to occur in the cells treated with a high concentration of the drug. These effects may be among the factors stimulating and/or accompanying high-dose mitoxantrone-induced programmed cell death or apoptosis.

    ID:796
  47. Feofanov A., Sharonov S., Kudelina I., Fleury F., Nabiev I. (1997). Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis. Biophys. J. 73 (6), 3317–3327 [+]

    Studying mechanisms of drug antitumor action is complicated by the lack of noninvasive methods enabling direct monitoring of the state and interactions of the drugs within intact viable cells. Here we present a confocal spectral imaging (CSI) technique as a method of overcoming this problem. We applied this method to the examination of localization and interactions of mitoxantrone (1, 4-dihydroxy-5, 8-bis-[([2-(2-hydroxyethyl)-amino]ethyl)amino]-9,10-anthracenedione dihydrochloride), a potent antitumor drug, in living K562 cells. A two-dimensional set of fluorescence spectra of mitoxantrone (MITOX) recorded with micron resolution within a drug-treated cell was analyzed to reveal formation of drug-target complexes and to create the maps of their intracellular distribution. The analysis was based on detailed in vitro modeling of drug-target (DNA, RNA, DNA topoisomerase II) interactions and environmental effects affecting drug fluorescence. MITOX exposed to aqueous intracellular environment, MITOX bound to hydrophobic cellular structures, complexes of MITOX with nucleic acids, as well as the naphtoquinoxaline metabolite of MITOX were simultaneously detected and mapped in K562 cells. These states and complexes are known to be immediately related to the antitumor action of the drug. The results obtained present a basis for the subsequent quantitative analysis of concentration and time-dependent accumulation of free and bound MITOX within different compartments of living cancer cells.

    ID:797
  48. Efremov R.G., Feofanov A.V., Dzhandzhugazyan K.N., Modyanov N.N., Nabiev I.R. (1990). Study of ATP binding in the active site of Na+,K(+)-ATPase as probed by ultraviolet resonance Raman spectroscopy. FEBS Lett. 260 (2), 257–260 [+]

    The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na+,K+-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH2 group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.

    ID:798