Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.2, #4)

Determination of the “Amino Acid Conflicts” and amino acid substitutions in primary structures of 41 human proteins by the proteomic technologies by M. A. Kovalyova; L. I. Kovalyov; L. S. Eryomina; A. A. Makarov; M. V. Burakova; I. Yu. Toropygin; M. V. Serebryakova; S. S. Shishkin; A. I. Archakov (325-334).
Proteomic studies of some human tissues and organs (skeletal muscles, myometrium, motor zone of the brain, prostate), and also cultivated myoblasts revealed 41 proteins, in which the presence of certain variants of amino acids (“conflicts”) was recognized at several “conflict” positions. Among the 93 registered “amino acid conflicts”, seven cases represented the results of the protein polymorphisms caused by corresponding substitution of individual amino acid. Proteomic analysis of prostate proteins revealed two isoforms of a prostate-specific antigen, formed due to alternative splicing. Thus, our results have shown that employment of the proteomic technologies may characterize various types of biochemical polymorphism in many human proteins.
Keywords: proteomics; human proteins; amino acid conflicts

Serum proteome profiling for diagnostics of ovarian cancer using ClinProt magnetic technique and MALDI-TOF mass spectrometry by R. H. Ziganshin; D. G. Alexeev; G. P. Arapidi; V. T. Ivanov; S. A. Moshkovskii; V. M. Govorun (335-342).
Using ClinProt magnetic beads with reverse-phase (MB-HIC 8 and HB-HIC 18), weak cation exchange (MB-WCX) and metal affinity (MB-IMAC Cu) surfaces fractions of peptides and proteins were isolated from human sera for their profiling by MALDI-TOF mass spectrometry. Proteome profiling of sera from basically healthy women (47 subjects, average age 49) and from women with verified ovarian cancer (stages 1-IV, 47 patients, average age 51) by means of MB-WCX beads allowed to generate the best diagnostic models based on Genetic Algorithm and Supervised Neural Network classifiers; these models demonstrated 100% sensitivity and specificity during analysis of the test set. Introduction of additional sera from patients with colorectal cancer (19) and ulcerous colitis (5) to the statistical model confirmed 100% ovarian cancer recognition. Statistical analysis of mass-spectrometry peak areas included to the diagnostic classifiers showed 3 peaks characteristic for ovarian cancer and 4 peak areas exhibiting changes associated with both ovarian and colorectal cancer.
Keywords: proteome profiling; serum; MALDI-TOF-mass-spectrometry; ClinProt magnetic beads; ovarian cancer

Modeling of ligands for native and chiral modified NMDA receptor NR1-binding core by A. S. Korotina; A. V. Dmitriev; V. A. Tverdislov (343-345).
Native and chiral modified (with non-enzymatic Asn racemization) NR1-ligand binding core of NMDA-receptor was modeled by means of molecular dynamic ligand modeling. Results demonstrated that Gly, D-Ser, D-Asn and D-Thr are ligands of the NR1-binding core of native NMDA-receptor, whereas the chiral modified NR1-binding core is characterized by the aliphatic non-polar amino acids D-Ala, D-Leu, D-Ile and D-Pro as ligands. These amino acids can be considered as effective ligands of the NMDA-receptor NR1-binding core in age-related pathology.
Keywords: NMDA-receptor; non-enzymatic racemization; ligands of NR1-binding core; molecular dynamics

Noninvasive insulin delivery systems by D. Kh. Zubaerova; N. I. Larionova (346-355).
The review considers commercial insulin formulations. Special attention is paid to difficulties and strategies of the development of alternative hormone delivery systems (buccal, transdermal, intranasal, pulmonary and oral). At the moment there is only one approved formulation of the noninvasive insulin in the world.
Keywords: insulin; formulations; delivery systems; pulmonary insulin; buccal insulin; peroral insulin

Genetically engineered insulin and its pharmaceutical analogues by D. A. Gusarov; V. D. Gusarova; D. I. Bayramashvili; A. F. Mironov (356-366).
Studies of replacement therapy of diabetes mellitus resulted not only in introduction of series of forms of insulin available at pharmaceutical market but also in new insulin analogues, which exhibit better control of blood glucose level. The present paper deals with basic tendencies in this field.
Keywords: genetically engineered human insulin; diabetes mellitus; insulin-aspart; insulin-glargin; insulin-detemir; insulin-lyspro

The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions by Yu. D. Ivanov; A. V. Ivanov; N. A. Petushkova; O. G. Gara; V. Yu. Kuznetsov; A. V. Podoplelov; A. I. Archakov (367-372).
The interactions between cytochrome P450 2B4 (d-2B4), NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the monomeric reconstituted P450 2B4-containing monooxygenase system in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of the optical biosensor IAsys+. Such mode immobilization was not accompanied by any loss of activities of the immobilized proteins. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (kon/koff) were (0.013 ± 0.005) × 106 M−1 s−1/0.05 ± 0.02 s−1, and dissociation constant (KD) was (0.26 ± 0.13) × 10−6 M. Comparison of kon, koff and KD values for d-Fp/d-2B4 complexes formed under hydroxylation (O-dealkylation) with corresponding constants obtained for the oxidized proteins of (0.10 ± 0.03) × 106 M−1 s−1/(0.14 ± 0.06) s−1, and (0.71 ± 0.37) × 10−6 M, respectively shows that the decrease in kon and an insignificant decrease in KD are associated with the increase of complex lifetime during transition from the oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in both hydroxylation conditions and in the case of oxidized forms of these proteins. In both cases formation of the ternary d-Fp/d-2B4/d-b5 complexes occurred.
Keywords: cytochrome P450 2B4; cytochrome b5 ; NADPH:cytochrome P450 reductase; optical biosensor; protein-protein interactions

The cholesterol lowering properties of the complex compound simvastatin with glycyrrhizic acid (simvaglyzin) in experimental models by V. A. Vavilin; N. F. Salakhutdinov; Yu. I. Ragino; N. E. Polyakov; M. B. Taraban; T. V. Leshina; E. M. Stakhneva; V. V. Lyakhovich; Yu. P. Nikitin; G. A. Tolstikov (373-380).
A molecular complex of simvastatin (SV) and glycyrrhizic acid (GA) (at their ratio of 1 : 4) has been synthesized. The complex named “simvaglyzin” (SVG) was stable in aqueous and aqueous-alcohol solutions at GA concentrations exceeding 0.2 mM. In vitro SVG acted as an uncompetitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA (3-HMG-CoA) reductase (Ki of 94 nM). Appearance of this inhibitory activity is associated with cytochrome P450-dependent conversion of SVG, because the addition of 1 mM metyrapone to the incubation medium fully prevented the inhibition of 3-HMG-CoA reductase. SV and SVG (used at 300 nM concentration) inhibited mevalonate synthesis rate by 39.15±8,27 and 38.85±3,04%, respectively. In vivo SVG showed a dose-dependent cholesterol lowering effect. In rats the cholesterol lowering effect of SVG used at daily doses equivalent to 66 and 100 mg/kg of SV was the same as the effect of SV administered at the daily dose of 200 mg/kg. The decrease in total cholesterol of blood serum was 7% and 9% (p < 0.05) and 8%, respectively. Myotoxicity of these SVG doses estimated by blood serum creatine phosphokinase (CPK) activity was lower than that of SV. In rats treated with SV the activity of CPK increased by 79% (p < 0.01), while in SVG treated rats it decreased by 30% and 36% (p < 0.05). Any increase of the hepatotoxicity markers alanine aminotransferase or aspartate aminotransferase in blood serum was not observed. The data suggest pharmacological synergism attributed to the SV-GA complex formation and increased safety of the resultant complex compared with a parent compound.
Keywords: simvaglyzin; simvastatin; glycyrrhyzic acid; 3-HMG-CoA-reductase inhibition; hypercholesterolemia in rats

Determination of protease activity in blood and microorganisms by L. V. Kozlov; A. M. Bichucher; A. A. Mishin; V. L. D’yakov; N. I. Leont’eva; N. M. Gracheva; L. I. Novikova (381-384).
A protease activity may be determined by means of immunoglobulins. Since proteolytic products apparently do not retain antigenic determinants of the initial substrate, the monitoring of enzymatic process may employ ELISA methods. The ELISA determination of functional activity of specific IgA1 protease has been used not only for detection of this enzyme, but also for measurement of its inhibition constants. IgG adsorbed onto a microplate was used for evaluation of total proteolytic activity. Varying pH values of the reaction medium it is possible to measure activity of neutral, alkaline and acid proteases. This approach was used for estimation total proteolytic activity of neutral proteases in blood serum. Due to high sensitivity of this method it was possible to dilute serum up to the level when serum inhibitors had not blocked enzyme activity. Assay of serum enzyme activity at acidic pH results in activation of pepsinogens and determination of pepsin activity. Measurement of a total level of serum pepsinogen activity may have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the detectable activity.
Keywords: proteases; immunoglobulins; IgA1 protease; activity; inhibition; ELISA

Molecular characteristics of the testosterone binding globulin (TeBG) were investigated in microaliquots of human serum samples using [3H]5-α-dihydrotestosterone radioligand assays. Under experimental conditions used TeBG demonstrated high conformational plasticity and ability to adopt three conformational states (oligo-, di-, and monomeric forms) with different functional activities. The blood TeBG parameters undergo significant changes in dependence of physiological conditions of an organism and also after negative external treatments, as shown in children living Chernobyl contaminated regions of Belarus. Preparations of a lipid polyene complex from the basidiomycete Laetiporus sulphureus exhibited in vitro high activity in correction of high affinity and positive cooperativity of TeBG interaction with its androgen ligand. This represents a basis for subsequent development of a new highly effective method for pharmacological correction of blood hormone transport characteristics altered under conditions of reproductive dysfunctions.
Keywords: testosterone binding globulin; lipid polyene complex of Laetiporus sulphureus

Oxidation and uptake of LDL by monocyte-derived macrophages from blood of patients with IHD by M. V. Bilenko; A. V. Khilchenko; N. A. Nikitina; D. V. Aksenov (393-405).
The main goal of this study was to test our hypothesis that monocyte-derived macrophages of patients with ischemic heart disease (IHD, MPIHD) are in vivo prestimulated (primed) or stimulated cells. Their capacity for LDL oxidation and uptake exceeds that of macrophages from healthy donors (MPN). Monocytes were isolated from blood of 18 healthy donors and 25 IHD patients and LDL preparations were obtained from plasma of 16 healthy donors (LDLN) and 15 patients with familial hypercholesterolemia (LDLH). Aerobic incubation of LDLN or LDLH with MPIHD resulted in earlier accumulation (by 1 h) of TBARS in LDL, earlier aggregation of LDL (1 h vs 3 h in the case of MPN), more pronounced LDL apoB fragmentation as well as increased LDL uptake with the increase in accumulation of total cholesterol (TCh; by 1.8–2.1-fold, p < 0.05–0.01) and the decrease in cell viability compared with MPN (p < 0.01). MPIHD and MPN exhibited more effective oxidation and uptake of LDLH than LDLN and in most tests this capacity (to oxidize and uptake LDL) increased under hypoxic conditions. These results demonstrate that macrophages of IHD patients are in vivo stimulated cells and that this stimulation, especially in combination with hypercholesterolemic LDL and local or generalized hypoxia, represent serious predisposition for onset or progression of atherosclerosis in IHD patients. The express test model based on MPIHD may be used for estimation of monocyte/macrophage stimulation in IHD patients as well as for optimization of drug therapy and screening new antiatherosclerotic and antiischemic drugs.
Keywords: monocyte-derived macrophages; LDL; ischemic heart disease; hypercholesterolemia; lipid peroxidation; atherosclerosis; hypoxia

Novel synthetic oxysterols (22S,23S)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one (I) and (22R,23R)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one (II) influenced biosynthesis of cholesteryl esters from [14C]acetate (85% and 180% of control at 5 μM concentration) in the human hepatoma Hep G2 cell line. Ketosterol (I) increased the level of cholesteryl ester biosynthesis from [14C]oleate in Hep G2 cells in a dose dependent manner, whereas the level of cholesteryl esters biosynthesis in the presence of ketosterol (II) reached the maximal value (269±20% of control) at 1 μM concentration of this compound. In a cell free system ketosterol (I) increased the rate of ACAT-dependent cholesterol acylation similar to 25-hydroxycholesterol, however, ketosterol (II)), efficiently stimulated an initial rate of ACAT-catalyzed cholesterol esterification, followed by rapid inactivation of this enzyme.
Keywords: oxysterols; cholesterol; cholesteryl esters; ACAT; Hep G2 cells

Attenuation of irreversible rat heart injury by reperfusion with metabolic protectors by O. I. Pisarenko; L. I. Serebryakova; O. V. Tskitishvili; I. M. Studneva (411-417).
The effects of intravenous infusion of potassium-magnesium aspartate (K-Mg-Asp), a glucoseinsulin-potassium cocktail (GIK), a combination of glucose, insulin and potassium aspartate (GIKAsp), and insulin (I) alone on metabolism of the risk area (AR) and cardiomyocyte membrane damage have been investigated in rats during reperfusion after myocardial regional ischemia. Acute myocardial infarction (MI) was induced by a 40-min occlusion of the anterior descending coronary artery followed by a 60-min reperfusion. During reperfusion, K-Mg-Asp, GIK, GIKAsp, I or the physiological solution (control) was infused into the jugular vein at a rate of 1 ml/kg/h. After reperfusion, the MI sizes were significantly lower than in control and reduced in the following order: K-Mg-Asp > GIKAsp > I > GIK. By the end of reperfusion with metabolic protectors, ATP and phosphocreatine levels in the AR were 2–2.5 times higher that in the control (56.3 ± 3.4 and 81.8 ± 7.9% of the initial values, respectively). The losses of aspartate and glutamate pool and lactate and glucose accumulation in AR were significantly lower in the experimental groups than in control. At the end of the reperfusion, the total creatine content in the AR decreased to 32.3 ± 2.3% of the initial value in control, but restored after perfusion with GIK, I and K-Mg-Asp to 78.0 ± 5.7, 76.7 ± 5.5, and 62.4 ± 5.6% (of the initial value), respectively. The recovery of most parameters of aerobic metabolism and cell membrane integrity was maximal in the GIK and I groups and insignificantly lower after reperfusion with K-Mg-Asp.The metabolic efficacy of these protectors corresponded to MI size limitation induced by their infusion. The results suggest that myocardial reperfusion with GIK, I and K-Mg-Asp is a promising adjunctive therapy in patients with acute MI.
Keywords: myocardial reperfusion; high energy phosphates; glutamate and aspartate; glucose and lactate; cardiomyocyte membranes; apoptosis; necrosis

Quantitative determination of metabolites in human urine by 1H NMR spectroscopy by T. N. Kolokolova; N. M. Sergeev; A. Yu. Korol’kov (418-425).
Conditions for registration of urinary 1H NMR spectra have been optimized in order to achieve maximal accuracy of quantitative analysis. Urinary samples from patients with acute pancreatitis have been investigated and spectral data of identified urinary metabolites and results of their quantitative determination are given. Employment of 1H NMR spectra is perspective for the development of new laboratory diagnostic methods.
Keywords: metabolites; urine; 1H NMR spectroscopy; acute pancreatitis

The antioxidant and peroxidase activities of saliva in patients with inflammatory periodontal diseases and possibility of their correction by I. V. Nikolaev; L. N. Kolobkova; E. O. Landesman; E. V. Stepanova; O. V. Koroleva (426-431).
The antioxidant and peroxidase activities of mixed saliva have been investigated during test loads in control group and patients with caries and combination of caries with inflammatory periodontal diseases. Antioxidant activity of saliva was significantly decreased in the last two groups and so its was corrected by means of xidiphone.
Keywords: periodontitis; gingivitis; antioxidants; peroxidase; xidiphone

Isatin is an endogenous indole, which is increased in mammalian brain and peripheral tissues under conditions of stress. Physiological concentrations of isatin inhibit natriuretic peptide (NPR) receptor binding and NPR-dependent signalling. The inhibition of NPR signalling by isatin is attenuated by a nonhydrolyzable ATP analogue. In this study we have demonstrated that short term incubation of rat brain synaptosomes with a physiological concentration of isatin caused a rapid 3-fold accumulation of ATP. The additional increase of ATP in the presence of tyrphostin, an inhibitor of tyrosine kinase, which uses ATP for phosphorylation of some proteins, suggests the dependence of this phenomenon on the activity of ATP-consuming systems. ATP inhibited binding of [3H]isatin to both particulate and soluble fractions of the rat brain. These results suggest that isatin induces accumulation of ATP, which in turn may displace isatin from both membrane-bound and soluble binding sites. Since natriuretic peptides are known to decrease stress hormone release this regulatory loop may be involved in the maintenance of natriuretic peptide signalling under conditions of stress and thus contribute to the control of stress responses.
Keywords: isatin; brain; ATP; natriuretic peptide receptors; stress