Laboratory of Biophysical Methods for Diagnostics

Staff

Staff

Head of the Laboratory
Gennady Dobretsov
corresponding member RAS,
Doctor of Physical and Mathematical
Sciences, Professor
Dobretsov

Vladimirov Y.A., member of RAS, Doctor of Biological Sciences, chief researcher
Mikhalchik E.V., Doctor of Biological Sciences, leading researcher,
Gularyan S.K., PhD, senior researcher
Smolina N.V., PhD, senior researcher
Syrejshchikova T.I., PhD, senior researcher
Ibragimova G.A., PhD, senior researcher
Svetlichny V.Y., researcher
Suprun M.V., researcher
Trakhtman I.E., researcher
Syromyatnikova E.D., junior researcher
Polyak B.M., junior researcher
Kalinina V.V., junior researcher
Isakova S.I., assistant researcher
Fedorkova M.V., assistant researcher

Topics of interest and research lines

Topics of interest and research lines

The major line of research in the laboratory is the development of diagnostic methods involving the use of fluorescent molecular probes.
This approach consists in the detection of changes in the physical properties of blood components, such as cells and proteins, during pathological processes. These changes are detected with specially synthesized small organic molecules, so-called molecular probes. Upon addition to blood, the probes bind specifically to a certain protein or blood cell, resulting in a significant change in shape and peak intensity in the fluorescence spectrum. These changes therewith depend on physical properties of proteins and cells. By using such fluorescent molecular probes, we succeeded in detecting the changes in the physical parameters of blood proteins and cells upon a number of pathological processes.

Main lines of research

Developing the theoretical foundation for fluorescent probe method.

Probes are highly sensitive to slightest changes in their immediate environment, in particular in the protein or lipid molecule bearing the probes. This property underlies their use. (Their use is based on this property). However, the complete description of the probe behavior as well as the interpretation of fluorescence data are impossible without the development of adequate physical models and computational methods. To use the probes for examining the spatial structure of membranes and lipoproteins, we needed to work out the mathematical models of nonradiative energy transfer between the donor (the bound probe) and the acceptor moiety in the protein molecule should be created (G.E. Dobretsov, O.V.Chekrigin, N.K.Kurek). This paved the way for the determination of the location of the apoprotein subunit within low density lipoproteins (LDL) (M.M. Spirin), very low-density lipoproteins (VLDL) (E.N. lapshin, N.K. Kurek), and discoidal high-density lipoproteins (HDL) (A.D. Dergunov).

The probe molecules bound to albumin, lipids, or even more complex objects, such as live cells, reside in the different environments and, hence, fluoresce differently. To clear up this heterogeneous situation, we used the technique for measuring fluorescence decay in the range of 10–11 –10–8 s.
Experiments performed with lipid membranes and lipoproteins let us to understand what happens in lipid molecules surrounding the probe (V.Yu. Svetlichnii).. The binding of probes to albumin occurs at several special sites. Fluorescence decay studies let us, for the first time, to succeed in observing the state of different centers simultaneously as well as in following up their changes during pathological processes (T.I. Sireitchikova, M.N. Komarova, Yu.A. Grizunov, N.V. Smolina).
However, to achieve this, we were constrained to develop a method for determining the concentration of fluorescing molecules through the use of the fluorescence decay parameters, and also the method for estimating the mobility of probe molecules in the heterogeneous medium such as the albumin molecule (G.E. Dobretsov, T.I. Sireitchikova) .
In recent years, large attention has been devoted to the understanding of physical reasons for the influence of surrounding medium on probe fluorescence. We conducted a series of model experiments including measurements of time-resolved transient absorption spectra in the time region of 10  13 s; and an analysis of the results was performed using Bakhshiev's theory of spectral shifts. The original results on the mechanisms of solvation of the probe molecule by surrounding medium were also obtained (S.K. Gularyan).
Of special note is the development of multiprocessing cluster for quantum chemical calculation of electronic structure and optical properties of probe molecules interacting with surrounding medium (S.K. Gularyan, B.M. Polyak). So high level calculations for fluorescent probes have been carried out for the first time. The recent results let us to understand why the probe possesses precisely this fluorescence and why it responds precisely so to surrounding medium. These questions with regard to fluorescence are known to be hard-to-solve ones.

lbfmd06Quantum chemical calculation reveals the causes of the change in fluorescence intensity of the probe 4-dimethylaminochalcone (DMC), accompanying a change in the polarity of the medium. An energy diagram shows low electron energy levels in the DMC molecule. GS is the electronic ground state, positioned to the upper of GS are the energy levels of the excited singlet and triplet state. In nonpolar media (for example, in lipid membranes or in lipoproteins), a transition from the singlet state 1 (p,p ) through 1 (p,p) to the triplet state leads to fluorescence quenching. By contrast, no fluorescence quenching occurs in polar media.

Albumin fluorescence-based test.

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Serum albumin acts as a transport protein for small organic molecules and has special binding sites for these ligands. Albumin is thus an active element in the mechanism of delivering of fatty acids, as an energy source, to muscle and toxic metabolites to detoxification organs, as well as it plays an important role in drug metabolism, and so on. Binding centers are known to undergo changes under pathological conditions. However, these data were not formerly used in clinical diagnostics. To study albumin binding centers, our group designed the fluorescent probe K-35 which, upon addition into blood, occupies all of the albumin binding sites (Fig.) (R.K. Aidiraliev,Yu.I. Miller, Yu.A. Grizunov). Practically all the fluorescence emission is therewith due to the probe molecules bound to albumin (M.N. Komarova, N.V. Smolina). The probe K-35 was synthesized in the laboratory of Prof. B.M. Krasovitsky at the Institute of Monocrystals (Ukraine, Kharkov).

To study the diagnostic capabilities of our approach in clinical trials, we designed and manufactured pilot lots of the reagent set. Studies carried out in more than 60 clinics established the efficiency of the test in prognosing at the early stage (the first 24 hours) the development of peritonitis (a multicenter study with Prof. A.A. Grinberg and Prof.G.V. Rodoman, RSMU, Prof. A.M. Federovsky, the I.M. Sechenov First Moscow State Medical University, Prof. V.G. Musselius, the N.V. Sklifosovsky Institute of Emergency Medicine, and others), in differential diagnosing, estimating the severity, and prognosing the development of acute pancreatitis (Prof. G.V. Rodoman, T.I Shalaeva, RSMU), and in prognosing the acute intoxication following the use of psychotropic medication following psychotropic medication ingestion (Prof.K.K. Il'iashenko, the N.V. Sklifosovsky Institute of Emergency Medicine, E.D. Syromyatnikova). Our test exceeded other laboratory blood parameters in this respect.
Interesting results were also obtained for stress and mental disorders (Prof. K.V. Sudakov, E.V. Koplik, Institute of Normal Physiology, RAMS, Prof. M.G. Uzbekov, E.Yu. Misionzhnik, Moscow Research Institute of Psychiatry, N.V. Smolina). This research line was supported by an international grant and by a grant from the Presidium of the Russian Academy of Sciences.
The albumin molecule contains a thiol group (SH) which can be oxidized/reduced and hence may influence on oxidative processes in blood. Studies of the changes of this group under pathological conditions are performed by our laboratory(N.V. Smolina, V.V. Kalinina, E.D. Siromyatnikova) in collaboration with the laboratory headed by Prof. O.A.Azizova. Preliminary results indicate the diagnostic significance of our test.

Search for diagnostically significant molecular parameters in blood plasma and wound exudate upon thermal burns.

Investigation and testing of cell membranes and blood lipoproteins.

Plasma level and properties of blood lipoproteins are known to correlate with the development of atherosclerosis and cardiovascular diseases (Fig.). Low- and very low-density lipoproteins (LDL and VLDL) are believed to be most atherogenic types of blood lipoproteins. Regular checking of LDL and VLDL levels is recommended for everyone by prevention programs for these diseases. Methods routinely used for measurement of LDL and VLDL are enzymatic assays. On initiative of Yu.M. Lopukhin, Full Member of the Academy of Sciences, our lab developed a fast fluorescence-based method for the direct measurement of these lipoproteins in micro-volume blood samples (E.N. Lapshin, N.K. Kurek, A.N. Ruchtin). Also, a clinical fluorometer and reagent sets were designed. The proposed method provides a quick (seconds) and inexpensive way to measure LDL and VLDL levels in blood and is suitable for population-wide screening.

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The method is based on the use of the special fluorescent probe K-37 (Prof. B.M. Krasovitsky, V.T. Skripkina ). This probe, after being added to blood, fluoresces strongly when it is located in (entrapped in) the hydrophobic, lipid core of lipoproteins.
The spatial structure of lipoproteins was examined using different variants of fluorescence probing method, such as nonradiative energy transfer between the probe molecules (M.M. Spirin, E.N. Lapshin, N.K. Kurek), and time-resolved fluorescence spectroscopy for measuring picosecond (namely 50 ps) fluorescence kinetics and time-resolved spectra (T.I. sireitchikova, M.N. Yakimenko, the Physical Faculty of MSU) . As a result, we determined the spatial structure of the protein moiety first in LDL and VLDL (M.M. Spirin, E.N. Lapshin, N.K. Kurek), then in discoidal high-density lipoproteins (A.D. dergunov) as well as in multiply modified LDL which are considered to play an important role in the development of atherosclerosis (A.S. Orekhov, O.M. Panasenko. S.K. Gularyan).

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An attempt was made to apply fluorescent probe method for detection of lipoprotein-like particles in leukocytes (S.K. Gularyan). The data obtained indicate the possible presence of such particles in granulocytes, but not in lymphocytes. The study was conducted, in collaboration with the Institute of Chemical Physics of the Russian Academy of Sciences (Prof. O.M. Sarkisov ), using two-photon excitation fluorescent microscopy. This technigues allows the detection of spectra and picosecond fluorescence decay at any point in a living cell. Figure shows a living HeLa cell stained with the fluorescent probe DMC. The DMC molecule binds to lipid structures. On the left is a fluorescent image of the cell. Fluorescence spectra and fluorescence decay kinetics were measured for each of 0.4  0.4 m2 parts of the image area. The physical structure appeared to be different in different cell regions. On the right in the figure, each color in the image corresponds to the cluster with a certain structural organization of lipid-protein complexes, as assessed by the combined evaluation of the parameters “spectrum+ decay” (S.K. Gularyan, V.Yu. Svetlichny). This approach may be used for monitoring changes occurring in cellular lipid structured upon pathological processes.

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Electric fields in blood cells and their changes upon pathological processes.

Using fluorescent probes, we measured simultaneously, for the first time, both plasma membrane potential and mitochondrial membrane potential, in lymphocytes (G.I.Morozova, V.V.Kosnikov). The results are presented in Fig.
Further research showed that membrane potentials are highly sensitive to allergic diseases, and, hence, it is possible to test allergens by measuring these potentials in patient’s lymphocytes (V.V. Kosnikov, Prof. V.A. Fradkin, the I.I.Mechnikov Research Institute of Vaccine and Sera). At that, in contrast to allergy skin testing, fluorescence-based analysis is performed in blood out of the body.
Membrane potentials in lymphocytes were found to change in response to bronchial asthma (Prof. N.A. Didkovsky , RIPCM ). These potentials also responds to tumor-associated antigens and to Tactivin, a therapeutic immunomodulator (Prof. V.Ya. Arion, O.V. Belova).
Thus, the measurement of membrane potentials in blood cells is a new source of information on the state of immunocompetent cells under immunological diseases. Given this, it is important to further develop this approach.

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Development of biochemical and biophysical methods to evaluate the state of hair and to diagnose hair

The aim of research is the development of biochemical and biophysical methods to evaluate the state of hair and to diagnose hair.
Human hair is a unique mini-organ. It consists of a follicle and a shaft. Cells of the follicle synthesize the proteins that form hair. With respect to the capacity for protein synthesis, the hair follicle takes one of the first places in the body.
Synthesis of the proteins keratins by hair follicle cells as well as fission and metabolism of the latter depend on energy supply.
ATP is the main energy source inside cells, but also it is an important regulator of processes occurring in hair follicles. Deviations from the normal ATP content may alter the hair growth cycle, reduce the active growth phase, and cause untimely hair loss (alopecia).

A hair shaft is influenced by various environmental factors. It can be damaged by ultraviolet radiation, sorbing metal ions, perming, and bleaching. In damaged hair, the permeability of a cuticle, a protective outer layer of the hair shaft, becomes altered, and an amount of labile proteins in a cortex, a middle layer, increases. It is well known that hair after perming or bleaching tends to release proteins.

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The figure conventionally illustrates the proteins of the hair shaft: colored in orange and green are light keratins which are water-soluble. Light keratins can covalently bind to heavy keratins. When protein-protein bonds (e.g. disulfide bridges (-S-S-)) are broken, an amount of free soluble proteins in the hair shaft increases. When hair gets in water, a part of light keratins (orange) stays within the hair shaft and another part (green) penetrates through the cuticle into the water. Proteins eluted in water can be measured by the corresponding biophysical and biochemical methods.

We attempted to answer the following questions:
- is it possible to measure hair bulb shealth ATP content in extracted hair?
- is their a difference in the ATP content between the patients with hair loss and healthy volunteers as well as between different areas of the hairy part of the head?
- is their a difference in the amount of labile proteins in hair between the patients with hair loss and healthy volunteers as well as between different areas of the hairy part of the head?

We compared two forms of alopecia, androgen-dependent hair loss and telogen hair loss. The more marked changes in the value of the measured parameters were observed among patients with androgen-dependent alopecia. For example, most of them exhibited a significant difference in the ATP content between the back and the crown of the head. Also, they exhibited the higher protein release for the back of the head as compared to healthy donors or the crown of the head.
For analysis to be performed, only five extracted hairs from the head area under examination are needed. The analysis resulted in the estimation of the average rate of protein release under standard experimental conditions, with the possibility of comparing it with the values determined for healthy individuals. Based on the results, one may draw a conclusion about the extent of hair damage and about recommendations on protective and reinforcing measures.

Main scientific achievements

Main scientific achievements

The work on the use of fluorescent probes for investigating biological membranes under normal and pathological conditions was begun by Prof. Valdimirov, Yu.A. at the Second Moscow Medical Institute in 1969, and was continued at RSI PCM from the moment of its establishment. For these years, we designed original probes, developed the theoretical background and methods for their use, and obtained clinically important results. Our results are not worse than those obtained by any other laboratory working in the field of techniques of fluorescent probes. We solved a number of theoretical problems that influence the development of this scientific field.
The probe molecule, which is about 1 nm in diameter, binds to proteins and lipids, thereby becoming a physical transducer, of the smallest possible size, that is sensitive to the structure of the surrounding protein and lipid moieties.
In 1983-90s, which coincided with the initial period of our institute and laboratory activity, preventing and controlling cardiovascular diseases which are causes of 50 % of deaths were declared an urgent task. One of the most important elements in disease prevention programs is regular checking of atherogenic lipoprotein levels. In the scale of the country, it implies 300 million analyses annually. The country did not possess technologies for all-round monitoring of population with respect to this analysis. Consequently, on the initiative of Lopukhin, Yu.M., the director of the institute, the Full Member of the Russian Academy of Sciences, our laboratory developed an original micromethod, having no analoques, for measuring atherogenic lipoproteins by fluorescence probing. The method allows screening of population and controlling of heart disease risk factors (E.N. Lapshin, N.K. Kurek, A.N. Rukhtin, R.K. Aĭdyraliev, Yu.A. Gryzunov). For large-scale clinical application of our method, we designed and manufactured a clinical fluorometer “ZOND AKL-01” (A.B.Bonokin, the V.A.Degtyarev Plant ) and reagent kits (G.V. Belevich, the Latvian Institute of Organic Synthesis), which were recommended for clinical use by Ministry of Public Health. In subsequent years, by using nanosecond fluorescence measurements, we achieved a more detailed determination of lipoproteins in a microamount of blood serum (T.I. Syrejshchikova).

Recently, the focus of our studies on lipoproteins and membranes is shifted to the level of the cell and subcellular organelles up to detection of a single molecule (S.K.Gularyan, V.Yu. Svetlichny, Prof. O.P.Sarkisov).
We determined the spatial localization of the apoproteins in low-density lipoproteins (M.M. Spirin, E.N. Lapshin, N.K. Kurek),in discoidal high-density lipoproteins (A.D. Dergunov, G.E. Dobretsov) as well as in multiply modified LDL which are characteristic of atherosclerosis (A.S.Orekhov, O.M. Panasenko, S.K. Gularyan). Also, elements of the tertiary structure of very low-density lipoproteins were determined.

Another line of our research is directed toward obtaining new information about physicochemical properties of blood proteins. These properties are altered under pathological processes. However, no suitable methods for assessment of these alterations and for use such information entered clinical practice. First of all, we studied changes in the properties of serum albumin, a transport protein in blood. To detect conformational changes in the albumin molecule, the fluorescent probe K-35 was synthesized (Prof. B.M. Krasovitsky, Institute for Single Crystals, Ukraine).
The clinical fluorescence-based test was developed to perform clinical investigation of diagnostic capabilities of this approach (R.K. Aĭdyraliev , Yu.I. Miller, Yu.A. Gryzunov). The reagent sets were designed, and pilot lots were manufactured (Yu.A. Gryzunov, A.B. Pestova, E.N. Kocajmani). Studies carried out in more than 60 clinics showed the effectiveness of the fluorescence-based albumin test in prognosing at the early stage the development of peritonitis (Prof. G.V. Rodom, T.I. Shalaeva), in differential diagnosing, estimating the severity and prognosing the development of acute pancreatitis (Prof. Uzbekov, E.Y. Misionzhnik, N.V.Smolina, E.V. Koplik), and in prognosing the acute intoxication following psychotropic medication ingestion (E.D. Siromyatnikova, Prof. K.K. Ilyashenko). The test was demonstrated to exceed, in this respect, other laboratory blood parameters. Interesting results were also obtained for stress and mental disorders (Prof. M.G. Uzbekov, E.Yu. Missionzhnik, N.V. Smolina, E.V. Koplik). This research line was supported by an international grant, a grant from RFBR, and a grant from the Presidium of the Russian Academy of Sciences.
An interesting and promising line of our research is based on the fluorescent methods to measure membrane potentials in a living cell. We proposed a new method, based on fluorescent probes, to measure simultaneously both plasma membrane potential and mitochondrial membrane potential, in lymphocytes (G.I. Morozova, V.V. Kosnikov). Further research (conducted together with clinics and colleagues from other laboratories) showed that the measurement of membrane potentials in lymphocytes may be used to test substances in searching for those provoking an allergic response in a patient. At that, in contrast to allergy skin testing, fluorescence-based analysis is performed in blood out of the body.

Changes in membrane potentials in lymphocytes were observed upon bronchial asthma (Prof. N.A. Didkovsky ). These potentials also respond to tumor-associated antigens (Prof. V.Ya. Arion, O.V. Belova).Thus, measurement of membrane potentials in blood cells is a new source of information on the state of immunocompetent cells under immunological diseases. Given this, it is important to further develop this approach.

Fluorescent probes demonstrated their potential in study of not only blood cells. Recently we started a work with other biological objects (E.V. Mikhalchik, N.V. Smolina).
Probes are highly sensitive to slightest changes in their immediate environment, in particular in the protein or lipid molecule bearing the probes. Designing of a new useful probe and a complete physical description of its behavior are a complicated challenge. In our laboratory, a group of new fluorescent probes (which may be used as a basis for new diagnostic methods) was designed. We were able to understand physical and physicochemical principles governing their behavior in blood and other biological objects (M.M. Spirin, E.N. Lapshin, N.K. Kurek, V.Yu. Svetlichny, S.K. Gulayan, Yu.A. Gryzunov, M.N. Komarova, N.V.Smolina, T.I. Syrejshchikova ). A new impulse to our work was given by the construction of our own workstation for quantum chemistry calculations of the excited states of probe molecules and the molecular dynamics of surrounding lipid molecules (S.K.Gularyan, B.M. Polyak ). These works were supported by grants from RFBR and the Russian Academy of Sciences, and by an international grant.

Publications

Publications

Books and reviews

1. Владимиров Ю.А., Добрецов Г.Е. Флуоресцентные зонды в исследовании биологических мембран. Москва: НАУКА, 1980, 320 с.
2. Добрецов Г.Е. Флуоресцентные зонды в исследовании клеток, мембран и липопротеинов. Москва: НАУКА, 1989. 277 с.
3. Kosnikov V.V., Dobretsov G.E. Electrical transmembrane fields in lymphocytes./ In: Physical Characterization of Biological Cells. Eds. W.Shutt et al. Verlag Gesundheit Gmbh, Berlin 1991, p.107-130
4. Альбумин сыворотки крови в клинической медицине. Под ред. Ю.А.Грызунова и Г.Е.Добрецова. Москва: Ириус, 1994, 226 с.
5. Альбумин сыворотки крови в клинической медицине. Книга 2. Под ред. Ю.А.Грызунова и Г.Е.Добрецова. Москва: ГЭОТАР, 1998 г., 440 с.
6. Лопухин Ю.М., Добрецов Г.Е., Грызунов Ю.А. Конформационные изменения молекулы альбумина: новый тип реакции на патологический процесс. Бюллетень Эксперим.Биол.и Мед., 2000, том 130, № 7, 4-8.
7. Добрецов Г.Е., Дергунов А.Д. Пространственная структура дискоидальных липопротеинов высокой плотности. Биологические мембраны, 2001, т. 18, № 5, 339-352
8. Лопухин Ю.М., Добрецов Г.Е., Владимиров Ю.А. Физико-химические методы анализа крови на основе флуоресцентных зондов.
Технологии живых систем, 2004, т.1, № 2, 29-36
9. Грызунов Ю.А., Закс И.О., Мороз В.В., Добрецов Г.Е., Комарова М.Н., Мещеряков Г.Н. Сывороточный альбумин: свойства, функции и их оценка при критических состояниях. Анестезиология и реаниматология, 2004, № 6, 68-74
10. Лопухин Ю.М., Добрецов Г.Е., Владимиров Ю.А. Создание новых физико-химических методов анализа крови на основе флюоресцентных зондов. Бюллетень Эксперим.Биологии и Медицины, 2007, Приложение 2, с. 43-48
11. Gryzunov Yu.A., Dobretsov G.E. Natural conformation of human serum albumin and its changes in pathology./ In: Protein Conformation: New Research/ Editor: L.B. Roswell. Nova Publishers, New York,. 2008, 125-159
12. Добрецов Г.Е., Грызунов Ю.А., Смолина Н.В., Родоман Г.В., Узбеков М.Г Альбуминовый флуоресцентный тест: результаты клинических испытаний (обзор). Эфферентная и физико-химическая медицина, 2009, т.1 № 1, 16-26
13. Добрецов Г.Е., Сырейщикова Т.И., Грызунов Ю.А., Смолина Н.В., Поляк Б.М., Бабушкина Т.А., Климова Т.П. Альбуминовый флуоресцентный тест: физико-химические основы (обзор). Эфферентная и физико-химическая медицина, 2010, т.2, № 2, 3-12
14. Добрецов Г.Е. Развитие технического арсенала метода флуоресцентных зондов. Обзор. Биофизика 2013, том 58, вып. 5, c. 741–747

Articles
1. Dobretsov G., Polyak B., Smolina N., Babushkina T., Syrejshchikova T.,.Klimova T, Sverbil V., Peregudov A., Gryzunov Y., Sarkisov O. Interaction of a fluorescent probe, CAPIDAN, with human serum albumin. Journal of Photochemistry and Photobiology, A: Chemistry, 2013, v.251, 134-140
2. Smolina N. V., Dobretsov G. E., Syrejshchikova T. I., Gamburg Yu. D., Kalinina V. V., Gryzunov Yu. A.. Nitrate Anion as a Probe for Electrostatic Interactions in Complexes Protein-Ligand. European Journal of Biophysics. 2013, v. 1, No. 2, pp. 22-27.
3. Михальчик Е.В., Н.В.Смолина, А.Г.Гаджигороева, Г.А.Ибрагимова, М.В.Федоркова , М.В.Супрун, Г.Е.Добрецов. Оценка количества слабосвязанных белков стержня волоса при алопеции. Клиническая дерматология и венерология, 2013, №3, 14-18.
4. Сакович Р. А., Б. М. Поляк, С. К. Гуларян, А. Н. Романов, В. Ю. Светличный, О.М. Саркисов. Квантово-химическое моделирование взаимодействия мембранного флуоресцентного зонда 4-диметиламинохалкона с гидроксильными группами окружения. Известия Академии наук. Серия химическая, 2013, № 5, 1142-1154.
5. Babushkina T. A., T. P. Klimova, A. S. Peregudov, Yu. A. Gryzunov, N. V. Smolina, G. E. Dobretsov, M. G. Uzbekov. Study of High-Resolution H1 Nuclear Magnetic Resonance Spectra of the Serum and Its Albumin Fraction in Patients with the First Schizophrenia Episode.
Bulletin of Experimental Biology and Medicine, 2012, Vol. 152, No. 6, 748-751.
6. Dobretsov GE, Syrejshchikova TI, Smolina NV, Uzbekov MG. Effects of fatty acids on human serum albumin binding centers. Bull Exp Biol Med. 2012 Jul;153(3):323-326.
7. Romanov A., Gularyan S., Polyak B., Sakovich R., Dobretsov G., Sarkisov O. Electronically excited states of membrane fluorescent probe 4-dimethylaminochalcone. Results of quantum chemical calculations. Physical Chemistry Chemical Physics, 2011, 13, No.20, 9518 – 9524.
8. Dobretsov G. E., Syreishchikova T. I., Smolina N. V. Molecular mobility of a fluorescent probe in binding sites of an albumin molecule. Biophysics, 2011, Vol. 56, No. 3, 403–406.
9. Syrejshchikova T.I., Gryzunov Yu.A., Smolina N.V., Komar A.A., Uzbekov M.G., Misionzhnik E.J., Maksimova N.M. Subnanosecond fluorescent spectroscopy of human serum albumin as a method for estimation of depression therapy efficiency. Laser Physics, 2010, v.20, No.5, pp. 1074-1078.
10. Dergunov A.D., Shabrova E.V., Dobretsov G.E. Composition, structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2010, v.A 75, 1100-1107
11. Gularyan S.K., V.Yu. Svetlichnyi, P.N. Zolotavin, G.E. Dobretsov, A.Yu. Kirillova, A.A. Astaf’ev, O.M. Sarkisov, N.G. Bakhshiev. Estimation of the Number of Polar Molecules in a Solvate Shell of a Fluorescent Probe 4-Dimethylaminochalcone. Optics and Spectroscopy, 2009, Vol. 106, No. 5, 660–665.
12. Михальчик Е.В., Питерская Ю.А., Липатова В.А., Пеньков Л.Ю., Ибрагимова Г.А, Коркина Л.Г. Активность антиоксидантных ферментов в ране при глубоких ожогах. Бюллетень Эксперим. Биол.мед. 2009, № 6, 696-699.
13. Михальчик Е.В., Питерская Ю.А., Будкевич Л.И., Пеньков Л.Ю., Факкиано А., К.Де Люка, Ибрагимова Г.А, Коркина Л.Г. Сравнительный анализ содержания цитокинов в плазме и раневом экссудате у детей с тяжелыми ожогами. Бюллетень Эксп. Биол.Мед. 2009, № 11. 524 -528.
14. Svetlichny V.Yu., Merola F., Dobretsov G.E., Gularyan S.K., Syrejshchikova T.I. Dipolar relaxation in a lipid bilayer detected by a fluorescent probe, 4’’-dimethylaminochalcone. Chemistry and Physics of Lipids 2007, 145, 13–26.
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