[INFLUENCE OF 29-40 AND 65-76 MCP-1 FRAGMENTS ON MYOCARDIUM MORPHOLOGY IN RATS AFTER T9CTHFMIA-R'FPFRFTTON].

Monocyte chemotactic protein-1 (MCP-1) is a chemokine that stimulates monocytes and macrophage migration into the sites of acute of chronic inflammation. Our study shows morphological changes in ischemic myocardium followed by the administration of two synthetic structural fragments of MCP-1 that are monocyte/macrophage migration inductor peptide IX and peptide X an inhibitor. Results show that peptides can change time points of the inflammatory response in myocardium. Peptide IX administration leads to increased and accelerated inflammatory response, i. e. attracts an additional number of monocytes and macrophages into the inflammatory focus. The introduction of the peptide X observed prolonged inflammatory process with the overall gain signs of myocardial damage.

[Study of neuroprotective, antihypoxic and antiamnesic effects of new mixture of tripeptides].

A new mixture of tripeptides (NMT: H-Lys-Asp-Glu-OH, H-Asp-Glu-Pro-OH, H-Asp-Glu-Arg-OH) in doses of 150 and 300 mg/kg per day produces clearly pronounced neuroprotective effect in rats with brain ischemia and decreases neurologic deficiency 1.1 times more effectively than reference drug semax. NMT (10, 50 and 150 mg/kg) had marked antihypoxic effect on mice in hermetic and altitude chamber. NMT in doses of 10 and 50 mg/kg was more effective than semax in hermetic chamber (1.3 and 1.5 times, respectively) and in a dose of 150 mg/kg in altitude chamber (1.9 times). NMT (50 and 150 mg/kg) had also marked antiamnesic effect on model amnesia caused by scopolamine in rats and was more effective (1.5 and 1.4 times, respectively) than semax in equal doses. NMT (50 and 150 mg/kg) also had marked antiamnesic effect on model amnesia caused by maximal electroshock and complex extreme factors in mice and in both doses was 4 times more effective than semax on the first model and in a dose of 150 mg/kg was 2.9 times more effective on the second model. NMT (50 mg/kg) increased the amplitude of transcallosal evoked potential in rat brain by 69% and was more effective than semax in equal dose. Thus, NMT is a promising neurotropic drug with neuroprotective, antihypoxic and antiamnesic activity.

[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.

[The immune-enzyme analysis based on chimeric molecule and oligopeptide fragmentations to detect autoantibodies to beta-adrenergic receptor in patients with dilation cardiomyopathy].

The article deals with specification of technique of immune-enzyme analysis to detect autoantibodies to beta-adrenergic receptors (beta1-AP) using compound of oligopeptids representing the fragmentations of extracellular sites beta1-AP and chimeric molecule of extracellular section of receptor This technique significantly exceeds the analogues defined in publications by its sensitivity and correlation with diagnosis.

[Novel conformational peptide antigen, which simulates an immunodominant epitope of the 2nd extracellular loop of ß1-adrenoceptor. Computer simulation, synthesis, spatial structure].

By means of computer simulation has been built polypeptide antigen conformational structure that imitates the immunodominant epitope of the 2nd extracellular loop of ß1-adrenoreceptor. A linear 25-membered peptide corresponding to calculated sequence was synthesized by means of solid-phase methoyd using Fmoc-technology, then directed by the closure ofdisulfide bridges was obtained original bicyclic polypeptide corresponding to the proposed structure of the conformational antigen. With the help of high-resolution NMR spectroscopy 3D structure of synthetic conformational antigen was investigated. It was shown that the structure of the bicyclic polypeptide similar to that of building computer model. Bicyclic conformational antigen was suitable for the detection of autoantibodies in the blood serum of patients with rhythm and conductivity violation without evidence of organic disease of the cardiovascular system.

[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.

[Suppression of vascular endothelium hyperpermeability by cell-permeating peptide inhibitors of myosin light chain kinase].

Novel peptides originating from the peptide inhibitor of myosin light chain kinase, L-PIK (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys), have been studied for ability to attenuate the thrombin-induced hyperpermeability of endothelial cell monolayer in culture. Peptides [NalphaMeArg1]-Lys-Lys-Tyr-Lys-Tyr-Arg-(D)Arg8-Lys and H-Arg(NO2)Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 (designated PIK2 and PIK4, respectively) appeared to be the most effective inhibitors of endothelial cell monolayer hyperpermebility, and surpassed other known peptide inhibitors of myosin light chain kinase derived from original L-PIK. Our results validate PIK2 and PIK4 as the leading molecules for the development of novel drugs intended to counteract pathological hyperpermeability of vascular endothelium.

[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).

[Affine sorbent with typtophil-threonil-tirosine for binding immunoglobulin G: sorption characteristics and aspects of practical application].

New chromatographic material based on tryptophil-threonil-tirosine was prepared. This sorbent effectively binds human, sheep, goat and cow immunoglobulins G. New sorbent shows high selectivity for removing immunoglobulins from blood plasma. Effective sorption capacity is 15-25 mg of immunoglobulin G per ml of matrix. Optimal method of covalent attachment ligand to polysaccharide matrix allows achieving high stability of the sorbents in terms of use and storage. This sorbent can be used in medicine and biotechnology.

[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.

[Peptide fragments of chemokine domain of fractalkine: effect on human monocyte migration].

Leukocyte chemotaxis to the area of tissue damage is mediated by chemokines. According to the primary structure, chemokines are divided into four families, fractalkine (CX3CL1) is the only one member of CX3C family and the only membrane-bound chemokine. Fractalkine molecule includes the extracellular N-terminal chemokine domain, mucin-like rod, the transmembrane and the intracellular domains. In membrane-bound state fractalkine has the properties of an adhesion molecule. Chemokine domain of fractalkine (CDF) is released from cell membrane by proteolysis, and this soluble form acts as a chemoattractant for leukocytes expressing fractalkine receptor CX3CR1. Fractalkine is involved in development of a number of pathological processes caused by inflammation, and therefore a search for fractalkine inhibitors is very important. For this purpose we identified several antigenic determinants--the fragments of CDF, and the following peptides were synthesized--P41-52 H-Leu-Glu-Thr-Arg-Gln-His-Arg-Leu-Phe-Cys-Ala-Asp-NH2, P53-60 H-Pro-Lys-Glu-Gln-Trp-Val-Lys-Asp-NH2 and P60-71 H-Asp-Ala-Met-Gln-His-Leu-Asp-Arg-Gln-Ala-Ala-Ala-NH2. The peptide effects on adhesion and migration of human peripheral blood monocytes expressing fractalkine receptors were investigated. In the presence of CDF and P41-52 we observed the increased adhesion and migration of monocytes compared with spontaneous values. Peptides P53-60 and P60-71 significantly inhibited monocyte adhesion and migration stimulated by CDF. Since the chemotactic activity of chemokines was shown to be dependent on their binding to glycosaminoglycans of the cell surface and extracellular matrix, the effect ofpeptides on the interaction of CDF with heparin was analyzed by ELISA. Peptide P41-52 competed with CDF for heparin binding, while peptides P53-60 and P60-71 had no significant activity.

Synthetic peptide fragment (65-76) of monocyte chemotactic protein-1 (MCP-1) inhibits MCP-1 binding to heparin and possesses anti-inflammatory activity in stable angina patients after coronary stenting.

OBJECTIVE AND DESIGN: The peptide from C-terminal domain of MCP-1 (Ingramon) has been shown to inhibit monocyte migration and possess anti-inflammatory activity in animal models of inflammation and post-angioplasty restenosis. Here, we investigate the effect of Ingramon treatment on blood levels of acute-phase reactants and chemokines in patients after coronary stenting and the mechanisms of Ingramon anti-inflammatory activity. SUBJECTS: Eighty-seven patients with ischemic heart disease (IHD) who faced the necessity of coronary angiography (CA) were enrolled. In 67 patients, one-stage coronary stenting was performed; 33 of them were treated with Ingramon in addition to standard therapy. Twenty patients underwent CA only. METHODS: High-sensitivity C-reactive protein (hsCRP) and fibrinogen blood levels were detected routinely. The chemokine concentration in plasma was measured by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array-based immunoassay. Intracellular Ca(2+) levels and cell surface integrin exposure were assayed by flow cytometry. MCP-1 dimerization was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). MCP-1-heparin binding was assessed with a biosensor and ELISA. RESULTS AND CONCLUSIONS: Ingramon treatment was accompanied by less pronounced elevation of hsCRP and fibrinogen levels and decreased MCP-1 concentration in plasma in patients after coronary stenting. Ingramon had no effect on MCP-1 interaction with cell receptors or MCP-1 dimerization, but inhibited MCP-1 binding to heparin. The anti-inflammatory activity of the peptide may be mediated by an impaired chemokine interaction with glycosaminoglycans.

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 the myelopeptides with differentiating activity].

The bone marrow myelopeptides Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro (MP-4) and Val-Asp-Pro-Pro (MP-6) have been synthesised by a classical method and by a solid phase synthesis. The differentiating activity of MP-4 and MP-6 in human leukemia cells HL-60 and K-562 has been studied. Both peptides induce terminal differentiation of these cell lines but the mechanism of action of peptides MP-4 and MP-6 is distinguished.

[Peptide inhibitors of myosin light chain kinase. Development of peptidase resistant analogues].

Myosin light chain kinase (MLCK) is the key regulator of various forms of cell motility including endothelial and epithelial permeability in particular. One of the potential MLCK inhibitors to be used in humans is a membrane permeable peptide H-RKKYKYRRK-NH2 (L-PIK). In present work we used solid phase peptide synthesis and Fmoc-technology to produce five modifications of L-PIK. Based on (1)H NMR analysis revealed that these peptides demonstrated improved resistance to degradation in blood plasma. One of de novo synthesized peptides, L-[MeArg(1)]PIK inhibited MLCK activity in vitro with the same efficiency as L-PIK whereas other modified peptides showed reduced inhibitory activity. D-amino acid analog of PIK was the least active inhibitor. Thus, we have demonstrated the possibility to produce an effective MLCK peptide inhibitor with increased resistance to biodegradation that is suitable for further pharmacological development.