Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.2, #1)
Biological activity of phytosterols and their derivatives by A. R. Mehtiev; A. Yu. Misharin (1-17).
This review is devoted to contemporary status of investigation of C-29 and C-28 plant sterols (phytosterols) in relation to their biological activity in mammals and mammalian cells. On the basis of experimental studies published during the last decade the following questions are discussed: phytosterols and nutrition; phytosterols and body cholesterol level; phytosterols and intestinal absorption of lipids, the role of phytosterols in lipid metabolism regulation; phytosterols and cultured mammalian cells; products of phytosterols oxidation; phytoecdysteroids and induced gene expression.
Keywords: sterols; phytosterols; cholesterol; lipid metabolism
Nitric oxide. Potentiation of NO-dependent activation of soluble guanylate cyclase: (Patho)physiological and pharmacotherapeutic importance by I. S. Severina (18-27).
The review highlights the molecular mechanism underlying the physiological effects of nitric oxide (NO), the role of signaling system: NO-soluble guanylate cyclase-cyclic 3′,5′-guanosine monophosphate (cGMP) in the realization of NO action. This review considers data on basic chemical characteristics of guanylate cyclase, such as the subunits structure, isoforms, modern concepts of the catalytic and regulatory centers of this enzyme. Realization of physiological effects of NO by guanylate cyclase depends on its heme prostetic group. NO-dependent activation of guanylate cyclase may be synergistically increased by a new NO-independent, allosteric activator of soluble guanylate cyclase-YC-1-(benzyl indasol derivative). Special attention is paid to the data on guanylate cyclase sites responcible for binding of the enzyme with YC-1 and the possible molecular mechanism underlying the synergistic increase of NO-dependent activation of soluble guanylate cyclase by YC-1. New compounds of endogenous nature capable to potentiate and synergistically increase the activation of guanylate cyclase by NO-donors have been found and investigated. The important physiological, pharmacotherapeutical and pathophysiological significance of this new fact is discussed.
Keywords: soluble guanylate cyclase; nitric oxide; YC-1; potenciation of activation
The methods of quantitative proteomics by A. T. Kopylov; V. G. Zgoda (28-46).
In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and funciotnal homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.
Keywords: quantitative proteomics; mass spectrometry; isotope label; chemical modification
Identification of human liver cytochromes P450 by using MALDI-TOF mass spectrometry by N. A. Petushkova; A. V. Lisitsa; I. I. Karuzina; V. G. Zgoda; G. F. Sheremetyeva; N. F. Samenkova; I. P. Nikitin; T. A. Sakharova; A. T. Kopylov; A. I. Archakov (47-54).
Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged.
Keywords: microsomes; liver; cytochromes P450; microsomal oxidation; one dimensional (1D) and two dimensional (2D) electrophoresis; mass spectrometry
Computer-aided design of polyketides with the required properties by A. P. Sergeyko; A. V. Stepanchikova; B. N. Sobolev; S. B. Zotchev; A. A. Lagunin; D. A. Filimonov; V. V. Poroikov (55-62).
An approach to rational design of new polyketides with the required spectrum of biological activity has been proposed. We have developed the BioGenPharm software, which generates combinatorial libraries of polyketides based on the user-defined input parameters, performs prediction of biological activity spectra for the generated structures and selection of molecuels with the required properties. PASS algorithm has been applied for prediction of polyketide activity spectra ( http://www.ibmc.msk.ru/PASS ). Validation of PASS prediction ability for polyketides was performed vs. the evaluation set containing 242 natural macrolides from the Dictionary of Natural Products. The mean prediction accuracy was 75.5%. The problem of choice of the cutting points for probability of the presence of activity (Pa), which provides optimal combination of such parameters as sensitivity, specificity, concordance has been considered. Applicability of the described method has been illustrated by generation of a virtual library of the erythromycin analogues and selection of substances with low probability of the hepatotoxic effect.
Keywords: polyketides; macrolides; virtual library; polyketide synthase; biological activity prediction; selection of substances with required properties
The role of PAR1 in protective action of activated protein C during nonimmune activation of mast cells by A. M. Makarova; L. R. Gorbacheva; T. S. Zamolodchikova; L. D. Rumsh; M. D. Smirnov; S. M. Strukova (63-70).
Activated protein C (APC) regulates the functional activity of mast cells by reducing release of β-hexosaminidase, the marker of mast cell degranulation. APC modulated not only spontaneous secretion from mast cells, but also secretion induced by the degranulators, proteinase-activated receptor agonist peptide (PAR1-AP) and compound 48/80. PAR1 desensitization by thrombin abolished the decrease of β-hexosaminidase secretion induced by low APC concentrations (≤1.5 nM). APC inactivated by phenylmethylsulfonyl fluoride (PMSF), did non mimic the enzyme action on mast cells. Duodenase (the duodenal proteinase) activated peritoneal mast cell via PAR1. APC abolished the proinflammatory effect of duodenase and PAR1-AP by reducing release of mast cell mediators. The effect of APC could be attributed to nitric oxide generation by mast cells because in the presence of L-NAME the secretory function restored. These data suggest involvement of mast cell PAR1 into regulatory mechanism responsible for the anti-inflammatory effect of APC.
Keywords: activated protein C; duodenase; proteinase activated receptors; mast cells; β-hexosaminidase secretion; nitric oxide; inflammation
Antirheumatic activity of methotrexate in phospholipid nanoparticles (Phosphogliv) by M. G. Zykova; V. N. Prozorovskii; O. M. Ipatova; T. I. Torkhovskaya; V. V. Glazatov (71-74).
The efficiency of methotrexate use in the basic therapy of rheumatoid arthritis is limited because of risk of side effects and fast drug efflux from zone of joints as well. We have developed a new stabilized form of methotrexate using phospholipid micelles of the injection form of the Phosphogliv preparation as a carrier. Phosphogliv has recently been developed in the Institute of Biomedical Chemistry (Moscow), as the emulsion of 50 nm phospholipid nanoparticles stabilized by glycyrrhizic acid. The conditions of maximal methotrexate incorporation into the phospholipid nanoparticles were optimized under control of HPLC (60% of total methotrexate was associated with nanoparticles). Methotrexate in phospholipid nanoparticles exhibited higher therapeutic efficiency in experimental adjuvant arthritis in rats than with free methotrexate. (This was evaluated by the decrease of edema and swelling of joints and inhibition of secondary inflammatory reaction.) The increase of antirheumatic activity of the developed preparation may also be attributed to the influence of glycyrrhizic acid, possessing both anti-inflammatory and immune properties. It is suggested to use a new form of methotrexate for intra-articular administration for rheumatoid arthritis treatment.
Keywords: rheumatoid arthritis; methotrexate; nanoparticles; phospholipids; phosphatidylcholine; glycyrrhizic acid
Substituted aminophenols and flavonoids as potential components for test-systems for total antioxidant activity by Yu. A. Grigorenko; E. I. Karaseva; D. I. Metelitza; V. L. Sorokin; G. A. Ksendsova; O. I. Shadyro (75-81).
Kinetics of ortho-phenylenediamine (PDA) oxidation has been investigated in the “pseudoperoxidase” system, Methemalbumin-H2O2, in the presence of 2-amino-4-tert-butylphenol (ATBP), 2-amino-4,6-ditert-butylphenol (ADTBP) and its four N-acyl derivates, as well as flavonoids (quercetin, morin, silibin, hesperidin and naringin). Under standard experimental conditions (20°C, phosphate buffered saline, pH 7.4, containing 5.25% ethanol and DMF) ATBP, ADTBP and two its N-acyl-derivatives and also all five flavonoids inhibited PDA oxidation with different efficiency, which was characterized in terms of the inhibition constants, Ki, M, or the percent of inhibition degree at the maximal concentrations of these inhibitors. Quercetin (Ki = 7.7 × 10−5 M) and ATBP (Ki = 1.26 × 10−4 M) were the most effective antioxidants. Using these characteristics and other required criteria, the pairs PDA-quercetin and PDA-ATBP were proposed for a practical application in the test-systems for evaluation of total antioxidant activity of biological objects.
Keywords: antioxidant activity; aminophenols; flavonoids; methemalbumin; ortho-phenylenediamine; pseudoperoxidase-catalyzed oxidation
Biodegradation of 125I-Labeled monoclonal antibodies after intravenous administration to rats with experimental C6 glioma by V. P. Chekhonin; K. A. Bulanov; G. M. Yusubalieva; E. A. Tsibulkina; O. I. Gurina; Yu. S. Skoblov; V. I. Shvets; Yu. A. Zhirkov; V. P. Baklaushev (82-87).
Employment of radiolabeled antibodies in biological studies, allows their specific accumulation in organs and tissues to be accurately detected. However, stability of such radiolabeled antibodies depends on both the method of labeling and particular experimental conditions. Therefore, stability of labeled antibodies should be determined in every particular experiment. In this study we have investigated stability of the 125I-labeled monoclonal antibodies to gliofibrillary acidic protein (GFAP), endothelial antigen AMVB1, and non-specific mouse IgG at the stage of their synthesis and after their intravenous administration to rats with experimental C6 glioma. Stability of labeled antibodies was determined in blood samples and homogenates of organs by the method of their precipitation with trichloroacetic acid. The 125I-radiolabeled antibodies were characterized by high radiochemical putiry, immunochemical activity and stability of the resultant preparations in blood and tissues, and the brain after administration in vivo. Electrophoretic analysis, thin-layer chromatography, and immunohistochemical tests have demonstrated the radiochemical purity, immunochemical competence, and stability of the labeled antibodies in vivo.
Keywords: C6 glioma; biodegradation; radiolabeled antibodies; monoclonal antibodies
Polyelectrolyte microcapsules as the systems for delivery of biologically active substances by T. N. Borodina; L. D. Rumsh; S. M. Kunizhev; G. B. Sukhorukov; G. N. Vorozhtsov; B. M. Feldman; E. A. Markvicheva (88-93).
Based on the method of the layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, sodium alginate (Alg) and poly-L-lysine (PLL), novel biodegradable microcapsules have been prepared for delivery of biological active substances (BAS). Porous spherical CaCO3 microparticles were used as templates. The template cores were coated with several layers of oppositely charged polyelectrolytes forming shell on the core surface. The core-shell microparticles were converted into hollow microcapsules by means of core dissolution with EDTA. Mild conditions for microcapsules preparation allow to perform incorporation of various biomolecules maintaining their bioactivity. Biocompatibility and biodegradability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme retained about 86% of the activity compared to a native chymotrypsin. The resultant microcapsules were stable in acidic medium and could be easily decomposed by trypsin treatment in slightly alkaline medium. Chymotrypsin was shown to be active after its release from the microcapsules decomposed by the trypsin treatment. Thus, the microcapsules prepared by the LbL technique can be used for the development of new type of BAS delivery systems in humans and animals.
Keywords: polyelectrolyte microcapsules; CaCO3 microparticles; chymotrypsin; proteolytic activity
Study of the binding of nuclear proteins from Plasmodium berghei strains with different chloroquine sensitivity to oligonucleotides corresponding to regulatory elements of the multidrug resistance gene by T. G. Pankova; T. M. Igonina; V. F. Kobzev; T. I. Merkulova (94-100).
Using electrophoretic mobility shift assay the proteins, specifically bound to the double-stranded oligonucleotides corresponding the binding sites of the transcription factors of AP1 family, NF-IL6 and SP1 involved in the up-regulation of human multidrug resistance (mdr1) gene and also proteins bound to the oligonucleotide corresponding to the element responsive for the stimulation by serum (SRE) have been found in the nuclear extracts of the rodent malaria parasite Plasmodium berghei (P. berghei). P. berghei strains exhibiting different chloroquine sensitivity are characterized by different patterns of nuclear protein binding to the oligonucleotides used. Mutations in the consensus sequences of AP1, NF-IL6, and SRE caused impairments in binding of some proteins; this suggests the existence of malaria parasite nuclear proteins containing DNA binding domains, which share similarity with corresponding DNA binding domains of transcription factors NF-IL6, SRF1 and the members of AP1 family. The results obtained suggest profound alterations in the regulatory apparatus of plasmodium during its selection for chloroquine resistance.
Keywords: P. berghei ; chloroquine resistance; transcription factors; oligodesoxyribonucleotides; electrophoretic mobility shift assay
Ubiquitin causes selective increase in the sensitivity of rat brain mitochondrial monoamine oxidases to various proteases by O. A. Buneeva; M. V. Medvedeva; A. E. Medvedev (101-104).
Incubation of rat brain mitochondria with ubiquitin and ATP followed by subsequent mitochondria sedimentation was accompanied by reduction of ubiquitin content in the supernatant. This decrease was more pronounced in the presence of ATP-regenerating system in the incubation medium (creatine phosphate/creatine phosphokinase). This ubiquitin incorporation into brain mitochondria observed only in the presence of ATP in the incubation medium increased sensitivity of monoamine oxidases (MAO) A and B to proteolytic inactivation by trypsin and papain, respectively. (Ubiquitin did not influence sensitivity of MAO B to trypsin and MAO A to papain). The data obtained suggest that ubiquitin incorporation into rat brain mitochondria increases susceptibility of MAOs to certain exogenous proteases, however, it remains unclear whether these changes stem from direct MAO-ubiquitin conjugation or reflect alterations in the membrane environment of these enzymes.
Keywords: monoamine oxidase; ubiquitin; proteolytic degradation; rat brain mitochondria; trypsin; papain
Distribution of mutations of acid β-D-glucosidase Gene (GBA) among 68 Russian patients with Gaucher’s disease by T. M. Boukina; I. V. Tsvetkova (105-110).
Gaucher disease (GD) is the most frequent lysosomal storage disease presenting in all populations. Mutations in the acid β-D-glucosidase gene (GBA) cause development of GD, resulting in a decrease or full loss of activity of this enzyme. We report here the results of the molecular-genetic analysis in 68 Russian GD patients from 65 families with the three types of this disease. The GD genotype has been completely elucidated in 58 patients and in all patients we have found at least one mutant allele (92.6%). Besides frequent mutations (p.N370S, c.1263_1317del (del55), p.L444P, p.R463C, Rec NciI) we have identified rare mutations p.R120W, p.R170C, p.R184W, p.G202R, Rec C (p.R120W; p.W184R; p.N188K; p.V191G; p.S196P; p.G202R; p.F213I), presenting in other populations of GD patients. The mutations p.P236T, p.L249Q, p.L288P, p.P319S, p.V352M, p.W381X, p.A384D identified in this study had not been described before. The GBA mutations identified in Russian patients have been compared with those found in patients of other European countries. Genotype-phenotype correlations in GD are discussed.
Keywords: Gaucher’s disease; acid β-D-glucosidase; acid β-D-glucosidase gene (GBA); molecular-genetic analysis; mutations
Functional activity of phagocytes of bronchoalveolar lavage in patients with fibro-cavernous and infiltrative pulmonary tuberculosis by M. E. Dyakova; O. T. Titarenko; D. S. Esmedlyaeva; A. V. Yelkin; N. P. Alexeyeva; T. L. Perova (111-114).
Functional activity of the bronchoalveolar lavage fluid (BALF) phagocytes was studied in 33 and 16 patients with fibro-cavernous and infiltrative pulmonary tuberculosis (FCPT and IPT, respectively). Complex examination of BALF, alveolar macrophages and neutrophils sedimented from BALF has shown interrelationship between functional activity of the cells and the form of pulmonary tuberculosis. Higher neopterin content and activity of elastase mainly secreted into BALF by activated alveolar macrophages and neutrophils, respectively, reflect higher secretory activity of both types of cells in FCPT. In FCPT this is combined with higher bactericidal activity of neutrophils, which significantly correlates with their adenosine deaminase (ADA) activity. Comparison of changes of the biochemical parameters studied in BALF (neopterin, elastase, ADA and its isoenzymes, 2-deoxy-ADA) and bactericidal activity of the sedimented cells obviously reflects different sides of BALF phagocytes functioning. Taking into consideration modern concepts on the mechanisms of regulation of phagocyte cells one may suggest the existence of differences in intercellular interactions in various forms of pulmonary tuberculosis.
Keywords: tuberculosis; neopterin; adenosine deaminase; NST-test; neutrophils macrophages