[Antioxidant properties of apelin-12 and its structural analogue in experimental ischemia and reperfusion].

Effects of apelin-12 H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH (A12) and its modified analogue H-(NMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH (I) on activity of antioxidant enzymes, formation of malonic dialdehyde (MDA) and generation of reactive oxygen species (ROS) were studied in ex vivo and in vivo models of myocardial ischemia and reperfusion (I/R) injury in Wistar rats. Preischemic infusion of peptide A12 or AI enhanced cardiac function recovery of isolated perfused heart and was accompanied by a marked attenuation of ROS generation detected by electron paramagnetic resonance (EPR) technique in myocardial effluent at early reperfusion compared with control. Intravenous administration (i.v.) of peptides in narcotized rats with regional myocardial ischemia limited infarct size and reduced activity of lactate dehydrogenase and MB-fraction of creatine kinase in plasma at the end of reperfusion. Treatment with peptide A12 prevented reduction or augmented activity of myocardial u/Zn superoxide dismutase, catalase and glutathione peroxidase by the end of reperfusion in both I/R models compared with control. Increased MDA content in the area at risk of rat heart in situ at the end of reperfusion was reduced to the initial value under the effect of i.v. A12 administration. Therefore, cardioprotective action of natural apelin-12 and its structural analog AI involve reduction of short-lived ROS generation and improvement of the antioxidant state of ischemic heart during reperfusion.

[Synthetic conformational antigen, simulating the extracellular part of M2-muscarinic receptor and its reactivity with sera from patients with idiopathic arrhythmias].

Linear peptides corresponding to fragment 83-98 of the first loop and fragments 168-192 and 171-182 of the second extracellular loops of M2-muscarinic receptor (marker of early cardiac disorders and arrhythmias) were synthesized by Fmoc-SPPS method. A new conformational antigen was synthesized by method of selective ligation of linear peptides by disulfide bond with native localization. Peptides were studied in reaction with sera from patients with idiopathic arrhythmias. A new conformational antigen was recognized by sera from patients with idiopathic arrhythmias with high reactivity.

[Synthesis and cardioprotective properties of apelin-12 and its structural analogs].

The apelin-12 and a number of its analogs, resistant to degradation of proteases, were synthesized by Fmoc- method of SPPS. By-products of synthesis were examined. It was found that serine hydroxyl group was sulfating during the final deprotection of apelin-12 (I) and its analogs. Sulfate moiety of Arg-protecting group transfer into hydroxyl group of Ser. Amount of by-product depends on presence of water in cleavage mixture. Furthermore, the final deprotection of amide analogs of apelin-12 (III, IV) is closed with formation of by-product--4-hydroxybenzylamide, its amount range on 20-8% on reaction mixture accordance HPLC data and also depend on composition of cleavage mixture. Effects of the synthesized peptides on recovery of cardiac function after ischemia were examined in a model of isolated perfused rat heart. Infusions of any of the peptides (I-V) before ischemia resulted in a significant improvement of contractile and pump function recovery compared to the control. Cardioptotective efficacy of the peptides increased in the following rank (I) < (II) = (III) < (IV) = (V).

[Involvement of NO-dependent mechanisms of apelin action in myocardial protection against ischemia/reperfusion damage].

Apelin 12 (A-12) was synthesized by the automatic solid phase method with the use of Fmoc technology. The synthesized peptide was purified by preparative HPLC and identified by 1H-NMR spectroscopy and mass spectrometry. Acute myocardial infarction was induced by 40-min LAD occlusion followed by 60-min reperfusion in narcotized Wistar rats. A-12 was administrated at the onset of the reperfusion at doses of 0.07, 0.35 and 0.70 micromole/kg; N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, was applied at a dose of 10 mg/kg 10 min prior to reperfusion alone or before A-12 administration (0.35 micromole/kg); saline was used in control. The indicated A-12 doses induced a transient reduction of the arterial systolic blood pressure (ASBP) to 85, 58, and 56% of the initial level, respectively, which was accompanied by its recovery by the end of reperfusion. All A-12 doses significantly limited myocardial infarct size by 26, 40 and 33%, respectively, compared to the value in control. After administration of A-12 at dose of 0.35 micromol/kg, this effect was combined with reduction of MB-creatine kinase (MB-CK) and lactate dehydrogenase (LDH) activities in plasma at the end of reperfusion by 56 and 47%, respectively, compared to the values in control. Inhibition of NO formation by L-NAME increased SABP but did not affect myocardial infarct size compared with that in control. Coadministration of L-NAME and A-12 resulted in lesser reduction of ASBP during reperfusion than injection of A-12 alone. This intervention led to an increase in infarct size by 26% with concomitant 1.8- and 1.5-times elevation of MB-CK and LDH activities, respectively, compared to the values in the A-12 group. The results indicate that NO is involved as a mediator of the effects of A-12 on the overall protection consisting in a limitation of infarct size and reduction of postischemic cardiomyocyte membrane damage. Cardioprotective mechanisms of apelin action are discussed.

[The influence of inhibiting no formation on metabolic recovery of ischemic rat heart by apelin-12].

Apelin 12 (A-12) was synthesized by the automatic solid phase method with use of Fmoc 1H-NMR spectroscopy and mass spectrometry. Effects of apelin-12 (a peptide comprised of 12 aminoacids, A-12) on recovery of energy metabolism and cardiac function were studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose that were subjected to global ischemia and reperfusion. A short-term infusion of microM 140 A-12 in KB prior to ischemia enhanced myocardial ATP, the total adenine nucleotide pool (SigmaAN = ATP + ADP + AMP) and the energy charge of cardiomyocites ((ATP + 0.5ADP)/SigmaAN) at the end of reperfusion compared with control (KB infusion) and reduced lactate content and lactate/pyruvate ratio in reperfused myocardium to the initial values. This effect was accompanied by improved recovery of coronary flow and cardiac function. Coadministration of 140 microM A-12 and 100 microM L-NAME (the nonspecific NOS inhibitor) profoundly attenuated the peptide influence on metabolic and functional recovery of reperfused hearts. The results indicate involvement of NO, formed under the peptide action, in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in postischemic heart.

In vivo reduction of reperfusion injury to the heart with apelin-12 peptide in rats.

Apelin-12 (A-12) peptide was synthesized by automated solid phase method and purified by reverse phase HPLC. Its homogeneity and structure were confirmed by HPLC, (1)H-NMR spectroscopy, and mass spectroscopy. Acute myocardial infarction was induced by 40-min occlusion of the left coronary artery with subsequent 60-min reperfusion in narcotized Wistar rats. Peptide A-12 was injected (intravenous bolus, 0.07 or 0.35 µmol/kg) to experimental animals simultaneously with the beginning of reperfusion. Injections of A-12 in these doses led to reduction of systolic BP to 67 and 85% of the initial level, respectively, which was virtually restored completely by the end of reperfusion, and to a significant reduction of the infarction focus in the myocardium (by 21 and 34% in comparison with the control, respectively). Injection of A-12 in a dose of 0.35 µmol/kg led to reduction of plasma concentrations of necrosis markers in comparison with the control by the end of reperfusion: MB-creatine kinase by 56%, lactate dehydrogenase by 30%. The results attest to vasodilatory effects of A-12 under conditions of heart reperfusion in vivo; the peptide injected after local ischemia limits the myocardial infarction size and reduces damage to cardiomyocyte membrane.

[Effects of exogenous apelin-12 on functional and metabolic recovery of isolated rat heart after ischemia].

Apelin 12 (A 12) was synthesized by the automatic solid phase method with the use of Fmoc technology. The synthesized peptide was purified by preparative HPLC and identified by 1H NMR spectroscopy and mass spectrometry. Effects of A 12 were studied on isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose. The hearts were subjected to 35 min global ischemia followed by 30 min reperfusion. A short term infusion of A 12 in KB (35, 70, 140, 280, and 560 M) was applied prior to ischemia (A 12 I) or at onset of reperfusion (A 12 R). KB infusion without A 12 was used in control. A 12 infusion enhanced recovery of coronary flow, contractile and pump function during reperfusion with the largest augmentation of these indices in A 12 I group. Thus after infusion of 140 M A 12 recovery of coronary flow, the LVDP HR product and cardiac output were 92+/-5, 81+/-5, and 77+/-5% of the initial values, respectively, in A 12 I group, 83+/-6, 61+/-5, and 52+/-5% in A 12 R group, and 76+/-2, 42+/-2, 32+/-2% in control by the end of reperfusion. Both A 12 groups exhibited significant reduction of ischemia/reperfusion contracture compared with control. Enhanced functional recovery in A 12 I group was combined with a decrease in lactate dehydrogenase leakage in perfusate at early reperfusion (at the average by 36+/-5% compared with control, <0.05). Preischemic infusion of 140 M A 12 markedly increased myocardial ATP content and twice decreased AMP accumulation at the end of reperfusion. These alterations resulted in enhanced preservation of the total adenine nucleotide pool (to 81+/-5% of the initial value vs. 66+/-3% in control, <0.05) and better recovery of the energy charge potential (0.77+/-0.01 vs. 0.60+/-0.06 in control, <0.005) in reperfused hearts. At the end of experiment myocardial lactate and lactate/pyruvate ratio were on average 5 fold lower in A 12 I treated hearts compared with control one and did not differ significantly from initial values. This finding implies that better restoration of energy metabolism in hearts protected with A 12 before ischemia might be attributed to ameliorated glucose oxidation during reperfusion. Therefore enhanced functional recovery of ischemic heart and lesser cell membrane damage induced by A 12 were associated with maintaining high energy phosphates, particularly ATP, in reperfused myocardium. Cardioprotective mechanisms of apelin action are discussed.

[Synthesis and properties of a new conformational antigen modeling an extracellular region of beta(1)-adrenoreceptor].

Two fragments corresponding to the 125-133 and 206-218 sequences of a molecule of the beta(1) adrenoreceptor (autoantibodies to this protein are often found in patients with dilated cardiomyopathy) were synthesized by the solid phase method with the use of Fmoc technology. Two new conformational antigens were prepared by directed (regioselective) and undirected (spontaneous) formation of intramolecular and intermolecular disulfide bridges between the corresponding cysteine residues of the synthesized peptides. One of these antigens consisted of a mixture of disulfide isomers, and another antigen was an isomer with a natural arrangement of S-S bridges. Immunosorbents were obtained by immobilization of the synthesizes antigens on the bromocyanogenactivated sepharose and applied to the removal of autoantibodies in a beta(1)-adrenoreceptor from the blood plasma of patients. We demonstrated that the sorbents on the basis of the conformational antigens were more effective in comparison with those containing linear peptide precursors.

[Effect of a cholecystokinin tetrapeptide analogue on opioid reception under acute and chronic morphine administration].

Effects of a modified CCK-4, a tetrapeptide fragment of cholecystokinin, on opioid reception and cAMP level were studied. The modified CCK-4 changed the ligand binding of the opioid receptors of mu- and sigma-types in vitro. In vivo, it prevented changes in opioid reception caused by a single morphine injection or by morphine withdrawal after its long-term introduction. The CCK-4 analogue did not exert any effect in the state of intoxication after a long-term introduction of morphine or even promoted the morphine effect. The introduction of the CCK-4 analogue alone or together with morphine changed the forskoline-stimulated level of cAMP. These changes depended on the brain structure and the duration of the introduction of morphine and the CCK-4 analogue. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

[Biological activity of cholecystokinin-(30-33) tetrapeptide analogs].

Analogues of the endogenous peptide corresponding to the 30-33 sequence of cholecystokinin (Trp-Met-Asp-Phe-NH2) were synthesized, and their biological activity was studied. It was shown that, in rats, the N-succinylated Nle2 analogue of this tetrapeptide exhibits increased anxiolytic properties in the dark-bright chamber test and an enhanced alcohol intake by both the control animals and the long-time alcohol-dependent animals under the conditions of free choice. Introduction of an isopropyl residue into the C-terminal amide of the Nle2 analogue resulted in the appearance of anxiolytic and antialcohol activity and the ability to increase the morphine analgesic effect in the tail-flick test on rats. The two synthesized analogues retained an affinity to cholecystokinin receptors.

Effect of synthetic peptide thrombin receptor agonist encapsulated in microparticles based on lactic and glycolic acid copolymer on healing of experimental skin wounds in mice.

PAR1 peptide thrombin receptor agonist (PAR1-AP) was encapsulated in microcorpuscles based on lactic and glycolic acid copolymer. The desorption profile of the preparation was studied in vitro and its wound-healing effects were studied on a model of cut skin wound in mice. The study showed that 90% PAR1-AP was desorbed over 6 h, but the peptide was detected in eluates from the microparticle surface after 23 h. The desorbed peptide retained its physiological activity and was capable of activating PAR1 receptors on human platelets. The study of the dynamics of experimental skin wound healing in mice showed lower number of macrophages in the wounds treated with PAR1-AP microparticles compared to the control (open wounds and wounds covered with microparticles) and higher number of fibroblasts on day 3 of tissue reparation. Hence, PAR1-AP desorbed from microparticles shortened the inflammation phase in the wound. On day 7 the best healing parameters were also observed in wounds treated with PAR1-AP microparticles, which attests to shortening of the proliferation phase and acceleration of wound healing.

[Methods of searching for antigenic determinants for proteins with known primary structure]. / Metody poiska antigennykh determinant dlia belkov s izvestnoi pervichnoi strukturoi.

Theoretical and experimental methods for locating antigenic determinants of proteins with known amino acid sequences are discussed. These methods are systematized on the basis of the theoretical approaches applied, and the efficiency of various predictive methods is compared. Some examples of experimental epitope determination for a number of proteins are given.

[Cardiolipin antibodies dependents on beta2-glycoprotein-1 in antiphospholipid syndrome]. / Zavisimye ot beta2-glikoproteina-1 antitela k kardiolipinu pri antifosfolipidnom sindrome.

AIM: To study beta2-GP-I-dependent binding of phospholipid antibodies (PAb) to phospholipids and this process participation in pathogenesis of antiphospholipid syndrome (APS). MATERIALS AND METHODS: IgG-fractions and sera from 20 patients with APS. Cofactor activity of beta2-GP-I isolated from serum of healthy donors was examined with modified immunoassay. RESULTS: Contrary to donor IgG, binding of IgG fractions isolated from sera of APS patients with cardiolipin grows dose-dependently in the presence of beta2-GP-I. Cofactor activity of beta2-GP-I is confirmed in the study of sera of APS patients. Sera containing beta2-GP-I-dependent antibodies to cardiolipin (aCL), unlike aCL-negative sera, react with solid-phase immobilized beta2-GP-I. CONCLUSION: It is confirmed that beta2-GP-I participates in interaction of PAb with cardiolipin. Pathogenetic implication of beta2-GP-I-dependent PAb for onset of APS is discussed.

[Synthesis and biological properties of artificial antigens based on 280-289 fragment of beta2-gpI]. / Sintez i biologicheskie svoistva iskusstvennykh antigenov na osnove peptida, sootvetstvuiushchego posledovatel'nosti 280-289 belka beta2-gpI.

A series of artificial antigens were synthesized on the basis of the FC(Acm)KNKEKKC(Acm)S peptide from the beta 2-glycoprotein I sequence: lipophilic analogues, the peptide-BSA conjugate, and multiple antigen peptide (MAP) containing eight copies of the peptide on an oligolysyl core. The solid phase method for acylation of the peptide with fatty acids and the HPLC analysis of the acylpeptides were described. Antigenic properties of the resulting compounds were evaluated by CL-ELISA.

[Synthesis and antigenic properties of peptide fragments of beta2-glycoprotein-I]. / Sintez i issledovanie antigennykh svoistv peptidnykh fragmentov beta2-glikoproteina-I.

A number of beta 2-glycoprotein-I peptide fragments were synthesized by using the Fmoc-scheme of the solid phase method. Antigenic properties of these peptides were determined by ELISA. Acm-protected FCKNKEKKCS peptide was shown to inhibit binding of anti-cardiolipin antibodies to cardiolipin.