[Antioxidative and detoxifying effects of analogues of delta-sleep inducing peptide (DSIP)].

16 DSIP analogues with substitutions of 1-2 amino acid residues were synthesized in order to investigate their potential use in medicine. Antioxidative properties of these peptides were studied in vitro and their detoxifying activity was examined in vivo on a model of toxicosis that was induced by the cisplatin cytostatic, which has been widely used in the cancer treatment. Practically all the studied DSIP analogues were shown to exhibit considerable direct antioxidative activity (AOA), and that of the ID-6 analogue was higher than AOA of DSIP and comparable with AOA of vitamin C and ß-carotine. This analogue also demonstrated the most pronounced detoxifying effect towards cisplatin action, resulting in a decrease in the animal death from the acute cisplatin toxicity to 17% (in comparison with 50-67% for the control animals) and restoration of a number of cisplatin-sensitive biochemical blood parameters: decrease in the activity of aspartate aminotransferase and alanine aminotransferase and downregulation of the concentration of the final products of nitrogen exchange (creatinine and urea). Thus, the DSIP-relative peptides could be promising agents for the decrease in the toxic effects of cytostatics that are used in oncology.

[Delta-sleep inducing peptide entrapment and release from polymer hydrogels based on modified polyvinyl alcohol in vitro].

The aim of the study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to evaluate peptide release kinetics from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels on the basis of poly(vinyl alcohol) acrylic derivative (Acr-PVA) as well as macroporous hydogels containing epoxy groups which were synthesized by copolymerization of this monomer with glycidyl methacrylate. The isotropic hydrogels were fabricated at positive temperatures while the macroporous hydrogels (cryogels) were prepared at the temperatures below zero. The peptide was entrapped into macroporous modified PVA hydrogels by addition of a peptide solution on previously fabricated matrices, while into PVA-GMA hydrogels containing epoxy groups peptide immobilization was carried out by incubation of hydrogel matrices in the peptide solution. In the case of isotropic hydrogels the peptide was added into the polymer mixture at a hydrogel formation reaction. The peptide release kinetics was studied by incubation of hydrogels in PBS (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by phase-reverse HPLC. DSIP release from the macroporous PVA hydrogel after 30 min incubation was 74, 70 and 64% in water, PBS and 0.9% NaCl, relatively, and it was completed in 3 hs. From the isotropic hydrogel the release neither peptide nor products of its degradation was not observed even after 48 hs of incubation. For freshly prepared hydrogel the release kinetics was as follows: 27 and 78% in 30 and 33 hs, relatively. In the case of the lyophilized hydrogel samples the peptide release was 63% in 30 min incubation while drying patterns at room temperature for 3 days resulted in significant peptide loss because its structure damage.

[Metabolic effects of delta-sleep inducing peptide during physiological aging of the organism].

Subcutaneous injection of delta-sleep inducing peptide (DSIP) to postnatal rats (aged from 2 to 24 months) during 5 consecutive days every months at a dose of 10 microg/100 g body weight favors normalization of the age-related changes in carbohydrate metabolism and shows hypoglycemic effect, as manifested by a decrease in the level of glycosylated hemoglobin in erythrocytes of test rats. The administration of DSIP in postnatal rats of different age also led to a decrease in serum total lipid level, total cholesterol level, and atherogenicity index and an increase in the level of high-density lipoprotein cholesterol.

[Effect of delta-sleep inducing peptide on the functional activity of some organs and tissues of rats during physiological aging].

The authors show that exogenous delta-sleep inducing peptide (DSIP) injected subcutaneously to the rats in the age of 2-24 months of postnatal development in a dose of 100 mg/kg of animal body weight in courses for 5 consecutive days every month, has a hepatoprotective effect. DSIP does not affect the functional activity of the pancreas, and is not involved in the regulation of calcium homeostasis in the physiological aging of the organism.

[Mechanism of delta-sleep inducing peptide geroprotective activity].

It is shown that the subcutaneous injection to the rats in the age from 2 to 24 months during 5 consecutive days every month with 10 microg/100 g body weight of delta-sleep inducing peptide (DSIP) suppresses lipid peroxidation preventing the increasing of malonic dialdehyde level in rats tissues and plasma, possesses a powerful antioxidant effect, which is realized by means of the activation of different endogenous antioxidant defense system of cell and extracellular fluid, including high- and low-molecular regulators of free radical processes. DSIP exerts stimulating influence upon the superoxid-dismutese, catalase, ceruloplasmin activities as well as the level of nonenzymatic antioxidants--urea and uric acids, because during organism aging the antioxidant defense systems are being suppressed. DSIP increases the volume of tissues and blood endogenous antioxidant defense system mainly by means of enzymatic antioxidant system, especially during later ontogenesis.

[Selective inhibitors of plasmepsin II from Plasmodium falciparum based on pepstatin].

A number of new inhibitors of plasmepsin II (PlmII) from Plasmodium falciparum, one of the key factors of malarial parasite survival, were synthesized. The inhibitors are analogues of pepstatin with various variants of Ala residue substitutions. Effects of the inhibitors on human PlmII and cathepsin D were studied. Inhibition of PlmII by the substrate was found, which required the use of the modified Henderson method for the determination of inhibition constants. Two synthesized inhibitors were shown to exhibit a pronounced selectivity to PlmII (K(i) = 5.5 and 5 nM) in comparison with cathepsin D (K(i) = 230 and 3000 nM, respectively).

Effect of delta sleep-inducing peptide on macromolecule biosynthesis in brain tissue of stressed rodents.

For evaluation of the nature of adaptogenic properties of delta sleep-inducing peptide we studied the effect of this substance on macromolecule biosynthesis in the brain of rats and mice exposed to burn injury and psychoemotional stress, respectively. Anabolic activity of delta sleep-inducing peptide depended on the purpose of adaptation corresponding to the type of stress.

[Biodegradable microparticles with immobilized peptide for wound healing].

Thrombin receptor agonist peptide (TRAP-6) may effectively replace thrombin for stimulation of damaged tissue regeneration. (Thrombin employment is limited by its high cost, instability and proinflammatory effect at high concentrations.) Immobilization of TRAP-6 into a poly(D,L)-lactide-co-glycolide (PLGA)-based matrix can protect peptides from a destruction by peptidases located in a wound area, and can also provide controlled release of the peptide. PLGA microparticles with immobilized peptide were produced by double emulsion/evaporation technique. An observation of microparticle morphology by scanning electron microscopy highlighted that peptide immobilization resulted in the increase of the microparticle porosity. TRAP-6 release kinetics was characterized by burst increase of TRAP-6 concentration in HEPES buffer solution (pH 7.5) for first 2 hours from the beginning of the experiment, and TRAP-6 complete release occurred for 20 hours. An investigation of TRAP-6 destruction by scanning electron microscopy revealed that the increase of microparticle size and surface porosity were observed already after 1 day of incubation in the buffer solution, and an aggregation of destructing microparticles was obvious by the 7th day of the incubation. Thus, peptide immobilization into PLGA microparticles can allow to develop a novel controlled release drug delivery system.

[Interaction of delta sleep-inducing peptide and its analogues with cellular membranes: a structure-function analysis].

The possibility of a correlation between the membrane properties of the delta sleep-inducing peptide (DSIP) and its analogues and their biological activity in vivo was examined by a comparative study of the membrane effects of these peptides. The peptides exhibiting biological activity in vivo were shown to cause a statistically reliable disordering of lipids in thrombocyte plasma membranes similar to the effect of DSIP. The membrane effect of the D-Val2, D-Tyr2, and Tyr1, Pro2 analogues of DSIP had the same bimodal dose dependence characteristic of natural DSIP. Only a slight nonspecific lipid disordering was registered for Trp-Asp-Ala-Ser-Gly-Glu, a biologically inactive hexapeptide analogue. These results indicate a correlation between the biological activity of the peptides during in vivo tests and their membrane properties in vitro. The structure-function relationship was studied within the group of DSIP analogues examined in vitro. The DSIP modeling effect, especially pronounced under the action of stress factors, was suggested to be directly associated with the ability of DSIP to change the dynamic structure of biological membranes.

Effect of calcium ions on enteropeptidase catalysis.

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.

Study of secondary specificity of enteropeptidase in comparison with trypsin.

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu)n-Lys(Arg)-(downward arrow)-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM(-1) x min(-1)) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM(-1) x min(-1)).

[Interaction of delta-sleep-inducing peptide with cell membranes in vitro]. / Vzaimodeistvie del'ta-son-indutsiruiushchego peptida s kletochnymi membranami in vitro.

The effect of delta-sleep-inducing peptide (DSIP) on erythrocytic membranes of human donor blood was studied by the spin label and spin probe methods. The spin-labeled derivative of DSIP containing the N-terminal residue of 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid was synthesized. An analysis of the ESR spectra of the spin-labeled DSIP derivative recorded after its incubation with a human erythrocyte suspension at 37 degrees C revealed a decrease in the rotational correlation time (tau c) and molecular order parameter (S) in comparison with the control solutions of the peptide in phosphate buffer (pH 7.4). The application of paramagnetic probes, 5-, 12-, and 16-doxylstearic acids and 3-doxylandrostanol, demonstrated that the introduction of DSIP in an erythrocytic suspension significantly increased the mobility of the hydrophobic area of the membrane bilayer both at a depth of 20-22 A and in the subsurface area (4-6 A). The dependence of these effects on the DSIP concentration was shown to have the form of a curve with well-defined extremes. The maximal disordering of membrane lipids was observed at peptide concentrations of 10(-9) and 10(-6) M. These results suggested that DSIP significantly affected the structure of plasmatic membranes in vitro by changing the physical state of their lipid components.

Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase.

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.

Effect of intranigral dosage with delta-sleep-inducing peptide and its analogs on movement and convulsive activity in rats.

Studies were carried out in rats on the effects of the administration of delta-sleep-inducing peptide (DSIP) and its analogs (1-4) into the reticular part of the substantia nigra on movement and convulsive activity. Intranigral microinjection of DSIP, and of DSIP-1 and DSIP-4, reduced horizontal and vertical movement activity as well as excursions to the center of the open field. DSIP, DSIP-2, and DSIP-3 had anticonvulsant effects, consisting of increases in the latent periods of the first convulsion and clonicotonic convulsions induced by picrotoxin, and reductions in the mean intensity of convulsions. It is suggested that changes in the structure of DSIP are accompanied by alterations in the strength of the effects of this peptide on horizontal and convulsive activity after dosage into the reticular part of the substantia nigra. The results indicating that these peptides have protective activity in experimental convulsive syndrome suggest that a relationship exists between DSIP-induced reductions in movement activity and the anticonvulsive efficacy of DSIP analogs when administered intranigrally, this being one of the components of the nigrodependent mechanisms of inhibition of convulsions.

Effects of delta-sleep inducing peptide (DSIP) and some analogues on the activity of monoamine oxidase type A in rat brain under hypoxia stress.

Metabolic effects of delta-sleep inducing peptide (DSIP) under hypoxia stress were investigated in rats subjected to short-term hypoxic conditions (about 0.26 Bar). It was found that DSIP partially restricted stress-induced changes in activity of mitochondrial monoamine oxidase type A (MAO-A) and serotonin level in rat brain. A number of DSIP analogues was tested and among them there were some compounds with enhanced ability to counteract hypoxia induced changes in MAO-A activity and serotonin content in comparison with native neuropeptide.

[Changes in the rate of protein biosynthesis in the organs of mice under the action of the delta sleep-inducing peptide and psychoemotional stress]. / Izmeneniia skorosti biosinteza belkov v organakh myshei pri deistvii del'ta-son-indutsiruiushchego peptida i psikhoémotsional'nogo stressa.

The effects of delta-sleep-inducing peptide (DSIP) and its analogs (ID-6 and ID-12) on the protein synthesis rate in the mouse brain, liver, and spleen were studied with special reference to mechanisms underlying the adaptogenic action of DSIP. Time-related changes of the protein synthesis rate were estimated in the mouse organs after a single intraperitoneal injection of the peptide (120 mg/kg body weight) and the psycho-emotional stress with or without preliminary (1 h before) injection of the peptide. After DSIP administration, the protein biosynthesis was activated and the dynamics of stress-induced changes of biosynthesis were modified. The data obtained suggest that the mechanisms underlying the DSIP adaptogenic action involve its modulatory effect on the regulatory system of protein biosynthesis.

[The effect of the intranigral use of the delta sleep-inducing peptide and its analogs on the motor and seizure activities of rats]. / Vliianie vnutrinigral'nogo primeneniia del'ta-son indutsiruiushchego peptida i ego analogov na dvigatel'nuiu i sudorozhnuiu aktivnost' krys.

The effects of delta-sleep inducing peptide (DSIP) and its analogues (1-4) administered into substantia nigra pars reticulata on locomotor and seizure activity were estimated in experiments in rats. It was shown that intranigral microinjection of DSIP as well as DSIP-1-DSIP-4 caused decreasing of horizontal, vertical locomotor activity and attendance of central sectors of the "open field". Antiseizure effects, i.e. the first and clonic-tonic picrotoxin-induced convulsive latent period lengthening and their intensity decreasing, revealed DSIP, DSIP-2 and DSIP-3. Authors suppose that changes of DSIP structure lead to changes of effects expression on locomotor and seizure activity in conditions of their administration into substantia nigra reticulata. Obtained data concerning protective effects of studied peptides on experimental seizure syndrome allow to conclude that there is interaction between DSIP-induced hypokinesia and DSIP analogues anticonvulsive effectiveness in case of their intranigral administration which is likelihood is one of the component of nigral-dependent seizure-suppressive mechanism.

[Synthesis and biological properties of delta-sleep peptide analogs. 2. Antimetastatic effect]. / Sintez i biologicheskie svoistva analogov peptida del'ta-sna. 2. Antimetastaticheskoe deistvie.

With the aim to investigate structure-functional relations of DSIP, 11 DSIP analogues were tested on antimetastatic activity, among them five new analogues, differing in positions 2 and 6 of the DSIP amino acid sequence were synthesized by the solid-phase method using Fmoc-approach. Experiments on C57B1 mice with metastatic Lewis lung carcinoma showed some analogues to be more efficient as antimetastatic agents then DSIP after i.v. (50 micrograms/kg) administration. Normalization of neuroendocrine status and activity of peritoneal, alveolar and spleen macrophages after the DSIP and some analogues injections in mice with metastatic Lewis lung carcinoma took place. Antimetastatic action of DSIP derivatives is considerably affected by structural changes, especially in the N-terminal part. Conformational factors rather than enhanced enzymatic resistance are essential for antimetastatic response.

[Changes in the intensity of free-radical reactions in the organs of rats under hypokinetic stress and with protection by the delta sleep-inducing peptide and its tyrosine-containing analog]. / Izmeneniia intensivnosti svobodnoradikal'nykh reaktsii v organakh krys pri gipokineticheskom stresse i zashchite del'ta-son-indutsiruiushchim peptidom i ego tirozinsoderzhashchim analogom.

Dynamics of the hypokinesia-induced changes of free radical and malonic dialdehyde content in brain, the changes of respiratory chain state and superoxide dismutase activity in liver of rats has been studied using ESR technique and biochemical methods. The effect of the preliminary injection of DSIP and its analogue ID-2 on these dynamics has also been studied. Using a model system constants of the interaction between DSIP or ID-2 and O2-. have been determined. The data point to the antioxidant effect of the administration of the peptides before hypokinesia. This suggests that the effect involves in mechanisms of the anti-stress action of these peptides.