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Purpose: Urokinase receptor (uPAR) promotes extracellular matrix proteolysis, regulates adhesion and cell migration, transduces intracellular signals through interactions with the lateral partners. The expression of uPAR and urokinase (uPA) is significantly upregulated in peripheral nerves after injury, however, little is known about uPAR function in nerve regeneration or the molecular mechanisms involved. The purpose of this study is to investigate the role of uPAR in nerve regeneration after traumatic injury of n. Peroneus communis in uPA-/-, uPAR-/- or control mice (WT) and in neuritogenesis in an in vitro Neuro 2A cell model., Results: Electrophysiological analysis indicates that nerve recovery is significantly impaired in uPAR-/- mice, but not in uPA-/- mice. These data correlate with the reduced amount of NF200-positive axons in regenerating nerves from uPAR-/- mice compared to uPA-/- or control mice. There is an increase in uPAR expression and remarkable colocalization of uPAR with α5 and β1 integrin in uPA-/- mice in recovering nerves, pointing to a potential link between uPAR and its lateral partner α5β1-integrin. Using an in vitro model of neuritogenesis and α325 blocking peptide, which abrogates uPAR-α5β1 interaction in Neuro 2A cells but has no effect on their function, we have further confirmed the significance of uPAR-α5β1 interaction., Conclusion: Taken together, we report evidence pointing to an important role of uPAR, rather than uPA, in peripheral nerve recovery and neuritogenesis., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest including any financial, personal or other relationships with other people or organizations., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(N α Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH 2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(N α Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH 2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Automated Fmoc solid-phase technique was used to synthesize Cys-containing linear peptide fragments of monocyte chemoattractant protein-1 and chemokine domain of fractalkine along with their analogues with Cys residue being either modified or replaced with Ser. Chimeric symmetric and asymmetric disulfides were also prepared from the former linear precursors. A SAR study on a set of the newly synthesized peptides revealed that capacity to stimulate migration of monocytes and to influence cell motility in vitro, in general, critically depends on the presence of Cys free thiol group in the molecule. Notably, all analogs lacking this feature, including chimeric disulfides, demonstrated lack of effect on monocyte migration.

Objective: To examine cardioprotective effects of Ρ-terminal fragment of adipokine apelin-12 (A12), its novel structural analogue [MeArg(1), NLe(10)]-A12 (I), and [d-Ala(12)]-A12 (II), a putative antagonist of APJ receptor, employing in vivo model of ischemia/reperfusion (I/R) injury., Materials and Methods: Peptides were synthesized by the automatic solid phase method using Fmoc technology. Anesthetized open-chest male Wistar rats were subjected to left anterior descending (LAD) coronary artery occlusion and coronary reperfusion. Hemodynamic variables and electrocardiogram (ECG) were monitored throughout the experiment. Myocardial injury was assessed by infarct size (IS), activity of necrosis markers in plasma, and metabolic state of the area at risk (AAR)., Results: Intravenous injection of A12, I, or II at the onset of reperfusion led to a transient reduction of the mean arterial pressure. A12 or I administration decreased the percent ratio of IS/AAR by 40% and 30%, respectively, compared with control animals which received saline. Both peptides improved preservation of high-energy phosphates, reduced lactate accumulation in the AAR, and lowered CK-MB and LDH activities in plasma at the end of reperfusion compared with these indices in control. Treatment with II did not significantly affect either the IS/AAR, % ratio, or activities of both markers of necrosis compared with control. The overall metabolic protection of the AAR in the treated groups increased in the following rank: II < A12 < I., Conclusions: The structural analogue of apelin-12 [MeArg(1), NLe(10)]-A12 may be a promising basis to create a new drug for the treatment of acute coronary syndrome.

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.

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.

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.

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.

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.

We have studied anti-tumor properties of bone marrow derived peptide Phe-Leu-Gly-Phe-Pro-Thr (MP-1) synthesized by classical methods. It was shown that MP-1 enhanced the effect ofcytostatic therapy of lymphatic leukemia P388 and increased latent growth period of P388 tumors implanted in irradiated mice. MP-1 also decreased metastasis of mouse breast adenocarcinoma Ca-755 after surgery.

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).

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.

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.

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.

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, &lt;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, &lt;0.05) and better recovery of the energy charge potential (0.77+/-0.01 vs. 0.60+/-0.06 in control, &lt;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.

A new anti-inflammatory peptide X (Ingramon)--a fragment of C-terminal sequence (65-76) of MCP-1, was elaborated. In Boyden chamber assay, Ingramon inhibited migration of human monocytic cells THP-1 and peripheral blood monocytes. In vitro in blood plasma, Ingramon was stable for at least 24 hours. Ingramon was accumulated in sites of inflammation in rats, and impaired monocyte and granulocyte accumulation occurred in different models of inflammation in rodents and primates. Ingramon exerted no effect on the MCP-1-dependent externalisation of p2 integrins CD lib CD18 (Mac-1). We propose that Ingramon might interfere with MCP-1-heparin/heparan sulphate binding on cell surface. Ingramon reception by volunteers showed its safety. The clinical trials of Ingramon in patients with acute coronary syndromes and percutaneous coronary interventions, have been launched.

Neuropeptide FF (H-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) injected intravenously temporarily enhanced the arterial pressure (AP) and the heart rate (HR). However, its role in the regulation of blood circulation is obscure. To study the properties of the molecule, its analogue was synthesized, in which proline in position 7 was substituted with glycine, and leucine in the position 2 with norleucine. Modified neuropeptide FF (FFm) also temporarily and in a dose-dependent manner increased the AP and HR; however, the equal degree of increase was reached at doses of FFm being 5-7 times lesser as compared with the natural peptide. The application of the FFm at hemorrhagic shock excluded mortality of animals during the experiment, considerably increased the degree of AP and HR restoration in the remaining experiments, and improved the survival of animals in 24 hours. It has been found that the level of antibodies to the fragment of hFF1 receptor in the serum is lower in spontaneously hypertensive rats SHR as compared with Wistar rats, but it is increased in patients of cardiological profile as compared with donors. The findings suggest involvement of neuropeptide FF in the regulation of blood circulation; however, the precise mechanisms remain to be determined.

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.

The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and an analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The biological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60-84% inhibition of growth of the following mouse cancers: lymphatic leukemia P-388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 toward T lymphocytes, but Ftc-MP-5 was practically inactive.

The retro-enantio analogue of peptide 66-77 of the chemokine MCP-1 and two hexapeptide fragments 66-71 and 72-77 of the C-terminal sequence of this protein were synthesized using the Fmoc strategy of solid phase peptide synthesis. The effect of the synthetic peptides upon the MCP-1-stimulated migration of THP-1 mononuclear cells was studied in vitro. The activity of the retro-enantio analogue was found to be comparable with that of the initial peptide 66-77: both peptides inhibit the migration of monocytes and granulocytes into inflammation zones of experimental animals.

The use of hydrogen peroxide for the formation of disulfide bridges was studied in 15 peptides of various lengths and structures. The oxidation of peptide thiols by hydrogen peroxide was shown to proceed under mild conditions without noticeable side reactions of Trp, Tyr, and Met residues. Yields of the corresponding cyclic disulfides were high and mostly exceeded those obtained with other oxidative agents, in particular, iodine. It was established that the use of hydrogen peroxide in organic medium also provided sufficiently high yields when large-scale syntheses of oxytocin and octreotide (up to 10 g) were carried out. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Potential antigenic determinants of the atypical lipoprotein-binding proteins T-cadherin (p105) and its precursor (p130) from cells of human smooth muscles were synthesized by the solid phase method according to the Fmoc-scheme. These corresponded to the 51-61, 140-160, 161-179, 260-271, 340-352, 350-362, and 370-385 sequences of p130 and were chosen on the basis of computer analysis of its antigenic structure. The conjugates of the peptides with horseradish peroxidase were used for the immunization of mice and rabbits. Antisera against the peptides corresponding to the 140-160, 161-179, and 260-271 sequences of p105 were shown by immunoblotting to react with p105, which we isolated from the vascular cells of smooth muscles and earlier identified as T-cadherin. These antisera inhibited the binding of low density lipoproteins with p105 in a dose-dependent manner. These results confirmed the identification of the p105 protein as T-cadherin and demonstrated the fundamental possibility of studying the interaction of this protein with low density lipoproteins by using antipeptide antibodies that inhibit binding.

The human immunodeficiency virus (HIV) proteins gp120 and gp41 are the principal immune target in HIV infection. One of the most important trends in the study of AIDS is linked to the mapping of sites involving in the binding to the cell receptor CD4 and in the induction of virus-neutralizing antibodies (VNA). Recent studies have revealed that gp120 as the major domain contains inducing type-specific BNA (PND) and a binding region with CD4 (CD4-BR). PND is located in the hypervariable loop of gp120 (residues 301-336 for a BRU strain), and CD4-BR is in the conservation area (residues 410-450). By using the synthetic fragments from these areas (BRU and MN strains) and HIV-infected persons' sera, the authors established that the immune response to PND and CD4-BR is somewhat interrelated: there is a synchronized response of HIV antibodies to peptides from the two regions in ELISA (r = 0.82). For analysis of this phenomenon, experiments with cross-linked immunoreactivity of rabbit antisera to peptides from PND and CD4-BR with homologous and heterologous peptides were performed by applying three control peptides from HIV and hepatitis B virus. It has been found that there is a cross reactivity between rabbit anti-PND (MN, BRU) and anti-CD4-BR abs. Peptide homological analysis revealed common structural elements for PND and CD4-BR despite significant differences in their proposed functions. There is a large amount of positively charged aa within both PND and CD4-BR which may be involved in gp120-CD4 interaction. Acetylation of Lys residues resulted in complete loss of peptide reactivity.

The amino acids involved in IgG reactivity to four HIV-1 gp41 overlapping synthetic peptides from the sequence 584-624 have been determined by a method based on the chemical modification of trifunctional amino acids, especially the acetylation of the amino groups of the lysine residues at pH 8-9. The reactivities of the sera from HIV-infected individuals and gp41-specific human Mab were studied with the overlapping peptides and their modified forms in indirect and competitive ELISA. Peptides 584-602 and 609-624 (CN-185) reacted with 88% of HIV-positive sera; the highest diagnostic significance (100%) was found with peptides 584-611 (AS-551) and 603-624 (CN-191). Acetylation resulted in a 10%-15% decrease in peptide reactivity. Moreover the concentration at which 50% inhibition occurred was 1.5 x 10(-6) M for unmodified AS-551 compared with 1.5 x 10(-5) M for the modified peptide. Circular dichroism spectra showed that acetylation did not alter the conformation of these peptides. Coupling of peptide AS-551 to a protein carrier at pH 6.5-7.0 did not affect the immunoreactivity of this peptide. Mab against human gp41 reacted with peptide 603-624 (CN-191). The concentration of this peptide necessary for 50% inhibition of Mab binding was 5.2 x 10(-6) M. It is concluded from the epitope mapping of the Mab that the antigenic determinant lies within the 603-609 fragment. Lys-608 appears to play a crucial role in the interaction with human HIVc-Mab.

alpha-, beta-, gamma- and (des-Tyr1)-gamma-endorphins were synthesised by classical methods of peptide chemistry. Optical purity of these peptides was controlled by GLC and NMR. Data on the opioid activity of the peptides synthesised are presented.