I. A. Grivennikov

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 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].

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.

Degradation of the ACTH(4-10) analog Semax in the presence of rat basal forebrain cell cultures and plasma membranes.

Summary.Here a new approach of the elucidation of paths of proteolytic biodegradation of physiologically active peptides, based on the use of a peptide with isotopic label at all amino acid residues and the enrichment of HPLC samples with unlabeled peptide fragments in UV-detectable concentration, has been proposed. The method has been applied for the investigation of degradation dynamics of the neuroactive heptapeptide MEHFPGP (Semax) in the presence of plasma membranes, and cultures of glial and neuronal cells obtained from the rat basal forebrain. The splitting away of ME and GP, and formation of pentapeptides are the predominant processes in the presence of all tested objects, whereas the difference in patterns of resulting peptide products for glial and neuronal cells has been detected. In conclusion, the approach applied allows analyzing physiologically active peptide concentrations in biological tissues and degradation pathways of peptides in the presence of targets of their action.

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.

Effects of Semax on dopaminergic and serotoninergic systems of the brain.

Short-term effects of the synthetic nootropic peptide Semax on the content and metabolism of monoamines in the brain of C57/Bl 6 mice and Sprague–Dawley rats were studied. Intraperitoneal injection of Semax at a dose of 0.15 mg/kg caused an increase in the tissue concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus and striatum of mice 0.5 and 2 h after the injection. Using brain microdialysis, we detected increased levels of extracellular 5-HIAA in the striatum of freely moving rats 1 h after administration of 0.15 or 0.6 mg/kg Semax. The effect lasted for an additional 3 h. These data suggest that Semax produces a modulatory effect on the serotoninergic systems of the animal brain, increasing the turnover rate of serotonin. The search for drugs improving memory and enhancing cognitive functions in humans, and the study of the mechanisms of their effects are an important scientific problem. Semax, a recently developed nootropic drug, is a synthetic peptide Met–Glu–His–Phe–Pro– Gly–Pro, structurally analogous to region 4–10 of adrenocorticotropic hormone (ACTH) but devoid of its hormonal activity [1]. The Pro–Gly–Pro segment of Semax is responsible for its metabolic stability and the relatively long duration of its nootropic effect. As shown in a number of studies, Semax improves learning and memory in animals, increases the concentration and attention during information processing, and relieves mental fatigue in humans [1, 2]. However, the neurochemical mechanisms of Semax effects are still far from clear. Recent studies demonstrated that various ACTH analogues, including Semax, possess the properties of antagonists of melanocortin (MC) receptors [3]. Convincing evidence has been provided for the existence of close functional and anatomical links between the melanocortin and monoaminergic systems of the brain [4, 5]. Intracerebroventricular administration of α -melanocyte-stimulating hormone induces grooming behavior in rats and simultaneously raises the intracellular level of dopamine in the striatum [6].

[The binding of Semax, ACTH 4-10 heptapeptide, to plasma membranes of the rat forebrain basal nuclei and its biodegradation].

The binding characteristics of the peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) to plasma membranes of basal nuclei of the rat forebrain and the dynamics of its degradation during its incubation with these membranes were studied. Binding of the homogeneously labeled [G-3H]Semax was shown to be time-dependent, specific, and reversible. Specific binding of the heptapeptide depended on calcium ions and was characterized by the dissociation constant of the ligand–receptor complex Kd 2.41 ± 1.02 × 10–9 M and by the concentration of binding sites Bmax 33.5 ± 7.9 × 10–15 mol/mg of protein. A method of studying Semax biodegradation in the presence of plasma membranes of rat brain was developed. It is based on the use of the peptide homogeneously labeled with tritium and on an HPLC analysis with UV detection at 220 and 254 nm of the peptide fragments formed. The half-life of Semax in the presence of the plasma membranes was demonstrated to be longer than 1 h. Dipeptidylaminopeptidases are considered to be the main enzymes responsible for its biodegradation; they successively cleave Semax to the HFPGP pentapeptide and the PGP tripeptide.

Expression of Regulatory GenesOct-4, Pax-6, Prox-1, and Ptx-2 at the Initial Stages of Differentiation of Embryonic Stem Cells in vitro

The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

The expression of HIV‐1 tat and nef genes induces cell‐specific changes in growth properties and morphology of different types of rat cells

Abstract.  Among the viral regulatory genes the tat and nef genes of HIV‐1 encode the proteins playing a central role in viral replication and exerting pleiotropic effects on the survival and growth of the cells. These effects differ in various cell types, possibly due to the use of genes from different HIV‐1 isolates. In this work, we studied the effects of the tat and nef genes on three types of cultured rat cells: primary embryo fibroblasts, pseudonormal Rat‐2, and pheochromocytoma PC12. Both genes affected growth properties and morphology of cells, the effects being cell‐specific. The proliferative activity of both Rat‐2 and PC12 cells was considerably increased after transfection with the tat gene. In primary rat embryo fibroblasts the tat gene induced multilayered foci. More importantly, it was shown that the efficiency of transformation was higher in cells coexpressing tat and nef. The nef gene caused considerable suppression of Rat‐2 cell proliferation, but no changes in their morphology. The nef gene transfection of PC12 cells also led to suppression of their proliferative activity. In addition, cellular agglomerates which were morphologically similar to multinuclear syncytial cells were detected in these cells for the first time.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.