N. F. Myasoedov

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium.

Summary. The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140–180°C was analyzed. This reaction was used for preparing [3H]enkephalins such as [3H]DALG with specific activity of 138 Ci/mmol and [3H]LENK with specific activity of 120 Ci/mmol at 180°C. The analogues of [3H]ACTG4–10 with specific activity of 80 Ci/mmol, [3H]zervamicin IIB with specific activity of 70 Ci/mmol and [3H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [3H]Peptide analysis using 3H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the β-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [3H] β-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.

Semax, an analog of ACTH(4-10) with cognitive effects, regulates BDNF and trkB expression in the rat hippocampus

The heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is an analog of the adrenocorticotropin fragment (4-10) which after intranasal application has profound effects on learning and exerts marked neuroprotective activities. Here, we found that a single application of Semax (50 microg/kg body weight) results in a maximal 1.4-fold increase of BDNF protein levels accompanying with 1.6-fold increase of trkB tyrosine phosporylation levels, and a 3-fold and a 2-fold increase of exon III BDNF and trkB mRNA levels, respectively, in the rat hippocampus. Semax-treated animals showed a distinct increase in the number of conditioned avoidance reactions. We suggest that Semax affects cognitive brain functions by modulating the expression and the activation of the hippocampal BDNF/trkB system.

The Effect of Semax and Its C-End Peptide PGP on the Morphology and Proliferative Activity of Rat Brain Cells During Experimental Ischemia: A Pilot Study

The neuropeptide preparation Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has been employed successfully in clinical practice for treating patients with severe brain blood circulation disorders. In spite of numerous studies, many aspects of the therapeutic effects of this preparation remain unknown. In this context, the effects of Semax and its C-end tripeptide PGP on the functional morphology of nervous tissue cells were studied in the normal rat brain and in a model of incomplete global rat brain ischemia. In control animals, both peptides activated the capillary network and caused similar morphological changes to neurons and the neuropil regions. We show here for the first time at the histological level that Semax and PGP increased proliferation of the neuroglia, blood vessel endothelium, and progenitor cells in the subventricular zone. In these experimental conditions, only Semax abated the manifestation of ischemic damage to the nervous tissue. This was probably attributable to a decrease in vascular stasis symptoms as well as the trophic effect of the peptide.

The Heptapeptide SEMAX stimulates BDNF Expression in Different Areas of the Rat Brain in vivo

N-terminal fragments of adrenocorticotropic hormone (ACTH) and different types ( α , β and γ ) of melanocyte-stimulating hormone (MSH) form the family of melanocortin peptides exerting a marked action on the functions of the central nervous system (CNS). Peptides from this family possess neurotrophic, nootropic and neuroprotective properties [1]. The heptapeptide SEMAX (Met–Glu–His–Phe–Pro–Gly–Pro) is an analog of the ACTH(4–10) fragment completely devoid of any hormonal activity present in the full-length ACTH molecule. It shown to stimulate the learning and memory formation processes in laboratory animals [2, 3]. As a regulator of CNS functions, this peptide, if administered at very small doses (15–50 μ g/kg), produces a marked nootropic effect [4, 5]. It also stimulates forebrain functions by increasing selective attention at the moment of information reception, improving memory consolidation and raising the learning ability [4]. At the cellular level, SEMAX has a neuroprotective effect, preventing the death of cholinergic neurons in culture, and stimulates an activity of choline acetyl transferase [6, 7].

Neurotrophin gene expression in rat brain under the action of Semax, an analogue of ACTH4-10

The heptapeptide Semax, an analogue of the N-terminal adrenocorticotropic hormone fragment (4-10) (ACTH(4-10)), has been shown to exert a number of neuroprotective effects. There are some investigations that connected these effects with the increase of neurotrophin gene expression under the peptide drug application in neuron cell cultures [M.I. Shadrina, O.V. Dolotov, I.A. Grivennikov, P.A. Slominsky, L.A. Andreeva, L.S. Inozemtseva, S.A. Limborska, N.F. Myasoedov, Rapid induction of neurotrophin mRNAs in rat glial cell cultures by Semax, an adrenocorticotropic hormone analogue, Neurosci. Lett. 308 (2001) (2) 115-118]. In this work, we examined the action of Semax on rapid changes of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) gene expression in vivo. Male Wistar rats were treated for 1h with Semax (50 microg/kg, single intranasal application) and neurotrophin gene expression in rat brain was analyzed by real-time polymerase chain reaction (PCR). It was revealed that an intranasal application of Semax increased the expression of both neurotrophin genes in rat hippocampus. Bdnf gene expression also increased in the brainstem and cerebellum. Ngf gene expression decreased in rat frontal cortex. Thus, Semax induces rapid, gene- and region-specific changes in neurotrophin gene expression in normal rat brain.

Disaccharide pyrimidine nucleosides and their derivatives: a novel group of cell-penetrating inhibitors of poly(ADP-ribose) polymerase 1.

Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3′-O-β-D-ribofuranosyl-5-iodo-dUrd (2d; IC50 = 45 μM), 3′-O-β-D-ribofuranosyl-2′-deoxythymidine (2e; IC50 = 38 μM), and 3′-O-β-D-ribofuranosyl-2′-deoxythymidine oxidized (4; IC50 = 25 μM). These compounds also reduced H2O2-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.

Semax, an analogue of adrenocorticotropin (4–10), binds specifically and increases levels of brain‐derived neurotrophic factor protein in rat basal forebrain

The heptapeptide Semax (Met‐Glu‐His‐Phe‐Pro‐Gly‐Pro) is an analogue of the N‐terminal fragment (4–10) of adrenocorticotropic hormone which, after intranasal application, has profound effects on learning and memory formation in rodents and humans, and also exerts marked neuroprotective effects. A clue to the molecular mechanism underlying this neurotropic action was recently given by the observation that Semax stimulates the synthesis of brain‐derived neurotrophic factor (BDNF), a potent modulator of synaptic plasticity, in astrocytes cultured from rat basal forebrain. In the present study, we investigated whether Semax affects BDNF levels in rat basal forebrain upon intranasal application of the peptide. In addition, we examined whether cell membranes isolated from this brain region contained binding sites for Semax. The binding of tritium‐labelled Semax was found to be time dependent, specific and reversible. Specific Semax binding required calcium ions and was characterized by a mean± SEM dissociation constant (KD) of 2.4 ± 1.0 nm and a BMAX value of 33.5 ± 7.9 fmol/mg protein. Sandwich immunoenzymatic analysis revealed that Semax applied intranasally at 50 and 250 µg/kg bodyweight resulted in a rapid increase in BDNF levels after 3 h in the basal forebrain, but not in the cerebellum. These results point to the presence of specific binding sites for Semax in the rat basal forebrain. In addition, these findings indicate that the cognitive effects exerted by Semax might be associated, at least in part, with increased BDNF protein levels in this brain region.

Effect of Semax peptide on survival of cultured rat pheochromocytoma cells during oxidative stress.

We studied the effects of Semax (antiinsulin peptide with neuroprotective effect) on the survival of cultured rat pheochromocytoma cell after oxidative stress induced by short-term incubation with hydrogen peroxide. Studies with fluorescent dyes propidium iodide and Hoechst 33258 showed that cell incubation with hydrogen peroxide led to the formation of damaged cells with characteristic signs of necrosis. Semax dose-dependently reduced the number of cells damaged by oxidative stress. The efficiency of Semax depended on the time of its addition to the culture medium. The results suggest that the neuroprotective effect of Semax in ischemic stroke can be due to its capacity to protect neurons from damage caused by oxidative stress.

Expression of inflammation-related genes in mouse spleen under tuftsin analog Selank

Previous studies have shown that synthetic tuftsin analogue Selank causes a transcriptomic response in the rat hippocampus and in spleen cells and may participate in the regulation of inflammatory processes in the body. In this work we studied the effect of Selank and two of its fragments on the expression of genes involved in processes of inflammation. We analyzed the expression of 84 genes involved in processes of inflammation (e.g., chemokines, cytokines, and its receptors) in mouse spleen 6 and 24 h after Selank single intraperitoneal injection (100 μg/kg) using real-time PCR method. We found significant changes in the expression of 34 genes involved in inflammation processes. The detailed analysis of quantitative data showed that the Bcl6 gene, which plays a main role in the formation and development of the immune system, exhibited significant changes in its expression levels in response to injection of each of the peptides. Also, we observed expression changes for Bcl6 target and corepressor genes under the influence of Selank and its fragments. Our results showed that Selank and its fragments caused a number of alterations in the expression of genes involved in inflammation. The data obtained confirmed the participation of Selank in the processes of regulation of inflammation in the body. The complex biological effect of Selank may be partially determined by the systematic effect of this peptide on genomic expression.

Efficiency of isotope exchange between sodium 4-phenylbenzoate and activated tritium

The efficiency of the protium–tritium isotope exchange in the sodium 4-phenylbenzoate (PBNa) molecule on activating the reaction on a tungsten filament at 1940 K (target temperature 77 and 295 K) and on heating the substrate supported on 5% Pd/C in the presence of gaseous tritium is compared. It is shown that the reaction mechanism is laregly determined by the properties of the material on which this reaction occurs and not only by the method of generation of activated tritium species. In the reaction of tritium atom with PBNa deposited on glass walls of the reaction vessel, the isotope substitution of tritium for protium occurred by the radical mechanism, leading to the formation of [3H]PBNa and hydrogenation products. It is assumed that the spillover of tritium atom over the support (carbon) surface is accompanied by polarization of the electronic shell and formation of the cluster (3+)(