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Ischemic stroke is an acute local decrease in cerebral blood flow due to a thrombus or embolus. Of particular importance is the study of the genetic systems that determine the mechanisms underlying the formation and maintenance of a therapeutic window (a time interval of up to 6 h after a stroke) when effective treatment can be provided. Here, we used a transient middle cerebral artery occlusion (tMCAO) model in rats to study two synthetic derivatives of adrenocorticotropic hormone (ACTH). The first was ACTH(4-7)PGP, which is known as Semax. It is actively used as a neuroprotective drug. The second was the ACTH(6-9)PGP peptide, which is elucidated as a prospective agent only. Using RNA-Seq analysis, we revealed hundreds of ischemia-related differentially expressed genes (DEGs), as well as 131 and 322 DEGs related to the first and second peptide at 4.5 h after tMCAO, respectively, in dorsolateral areas of the frontal cortex of rats. Furthermore, we showed that both Semax and ACTH(6-9)PGP can partially prevent changes in the immune- and neurosignaling-related gene expression profiles disturbed by the action of ischemia at 4.5 h after tMCAO. However, their different actions with regard to predominantly immune-related genes were also revealed. This study gives insight into how the transcriptome depends on the variation in the structure of the related peptides, and it is valuable from the standpoint of the development of measures for early post-stroke therapy.

This study investigated the effects of glyproline neuropeptide compounds (selank and Pro-Gly-Pro) on the white blood cell count and phagocytic activity of neutrophils in 40 nonlinear white male rats aged 68 months with experimental hyperthyroidism. Experimental hyperthyroidism was simulated by intragastric administration of L-thyroxine sodium salt pentahydrate at a dose of 150 g/kg/day for 21 days. All animals were equally divided into four groups: healthy rats (control), rats treated with L-thyroxine sodium pentahydrate (hyperthyroidism), rats treated with selank, and rats treated with Pro-Gly-Pro at doses of 200 g/kg/day intraperitoneally for 21 days, starting one day after the last administration of L-thyroxine sodium pentahydrate. After the animals were removed from the experiment, the white blood cell count and the percentage of lymphocytes, stick, and segmentonuclear neutrophils were calculated, and the phagocytic activity of neutrophils was evaluated. In the case of experimental hyperthyroidism, neutrophilic leukocytosis with a shift to the right, lymphopenia, and decreased phagocytic activity of neutrophils were observed. Glyproline neuropeptides contributed to the correction of observed changes in white blood cell indices and phagocytic processes, which indicates the immunocorrigating effect of the test compounds. Thus, glyproline neuropeptides demonstrated pronounced immunotropic activity, which manifested in the correction of changes arising from the leukocyte count and phagocytosis processes. However, further detailed study of the pharmacological effects of neuropeptide agents on experimental hyperthyroidism is necessary.

This study aimed to determine the peculiarities of behavioral reactions and psychoemotional status of laboratory animals in experimental hyperthyroidism conditions against the background of introducing peptides of glyproline series (selank and tripeptide Pro-Gly-Pro). All experiments were conducted on white non-linear male rats, which were divided into groups consisting of 10 individuals. The first group consisted of control rats that received intraperitoneal water for injection in equi-volume. The second group consisted of individuals with experimental hyperthyroidism modeled by intragastric administration of L-thyroxine Berlin-Chemi Germany) at 150 mcg/kg/day for 21 days. The third and fourth groups were animals with a hyperthyroidism model and received intraperitoneal Selank and Pro-Gly-Pro at 200 mcg/kg/day for 21 days after the hyperthyroidism model development. The behavioral activity determination of laboratory animals was conducted using the Open Field method, which is widely used in the analysis of approximate research activity, locomotive stereotyping, and the emotional reactivity level of rodents. The level of animal anxiety was assessed using the Raised cross-shaped maze method. The study revealed that experimental hyperthyroidism due to the introduction of L-thyroxine is accompanied by an increased level of the anxiety-depressive state in the behavior of male rats. The study on the effects of selank and its structural analog Pro-Gly-Pro on the psychoemotional status of male rats using Open Field and Raised cruciform labyrinth methods revealed that the recovery of the approximate research motor activity of laboratory animals is noted, which indicates the psychomodulatory activity of glyproline neuropeptides, in experimental hyperthyroidism conditions.

Objective. To investigate the effect of glyprolines on the levels of initiating and effector caspases in the serum of white rats under "social" stress. Materials and methods. The study was conducted on 90 white male rats of 6 months of age. All manipulations with animals were carried out in accordance with international and domestic requirements for working with laboratory animals. When modeling "social" stress, groups of animals with aggressive and submissive behavior were formed. Laboratory animals, taking into account the types of behavior, were divided into groups (n=10): a group of intact males (control); a group of animals exposed to" social " stress for 20 days (stress); groups of individuals who received intraperitoneal Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro), Pro-Gly-Pro, Pro-Gly-Pro-Leu at doses of 100 mcg/kg / day from the 1st day of stress exposure within a 20- day course. The effect of neuropeptides on the activity of apoptosis processes was evaluated by determining the level of initiating and effector caspases (caspase-8 and caspase-3) (ELISA Kit for Caspase-8 and ELISA Kit for Caspase-3; USA) in the blood serum of white rats by enzyme immunoassay. Results. According to the results of the study, it was found that under conditions of "social" stress, an increase in apoptotic processes was observed, accompanied by an increase in the level of caspase-3 and caspase-8 in the blood serum of white rats. The introduction of the studied compounds against the background of stress contributed to a decrease in the level of the studied indicators, which is most likely due to the presence of antiapoptotic action in glyprolins due to inhibition of the caspase-dependent cascade of apoptosis reactions, as a result of which the destruction of cellular structures occurs by hydrolysis of nuclear lamina, cleavage of adhesive proteins, destruction of the cytoskeleton. Conclusion. Thus, the conducted study established the presence of Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selank), Pro-Gly-Pro and Pro-Gly-Pro-Leu under conditions of stress-induced antiapoptotic activity due to inhibition of the caspase-dependent cascade of apoptosis reactions.

Glyprolines are Gly-Pro (GP)- or Pro-Gly (PG)-containing biogenic peptides. These peptides can act as neutrophil chemoattractants, or atheroprotective, anticoagulant, and neuroprotective agents. The Pro-Gly-Pro (PGP) tripeptide is an active factor of resistance to the biodegradation of peptide drugs. The synthetic Semax peptide, which includes Met-Glu-His-Phe (MEHF) fragments of adrenocorticotropic hormone and the C-terminal tripeptide PGP, serves as a neuroprotective drug for the treatment of ischemic stroke. Previously, we revealed that Semax mostly prevented the disruption of the gene expression pattern 24 h after a transient middle cerebral artery occlusion (tMCAO) in a rat brain model. The genes of this pattern were grouped into an inflammatory cluster (IC) and a neurotransmitter cluster (NC). Here, using real-time RT-PCR, the effect of other PGP-containing peptides, PGP and Pro-Gly-Pro-Leu (PGPL), on the expression of a number of genes in the IC and NC was studied 24 h after tMCAO. Both the PGP and PGPL peptides showed Semax-unlike effects, predominantly without changing gene expression 24 h after tMCAO. Moreover, there were IC genes (iL1b, iL6, and Socs3) for PGP, as well as IC (iL6, Ccl3, Socs3, and Fos) and NC genes (Cplx2, Neurod6, and Ptk2b) for PGPL, that significantly changed in expression levels after peptide administration compared to Semax treatment under tMCAO conditions. Furthermore, gene enrichment analysis was carried out, and a regulatory gene network was constructed. Thus, the spectra of the common and unique effects of the PGP, PGPL, and Semax peptides under ischemia–reperfusion were distinguished.

Ischemic stroke continues to be one of the leading causes of death and disability in the adult population worldwide. The currently used pharmacological methods for the treatment of ischemic stroke are not effective enough and require the search for new tools and approaches to identify therapeutic targets and potential neuroprotectors. Today, in the development of neuroprotective drugs for the treatment of stroke, special attention is paid to peptides. Namely, peptide action is aimed at blocking the cascade of pathological processes caused by a decrease in blood flow to the brain tissues. Different groups of peptides have therapeutic potential in ischemia. Among them are small interfering peptides that block protein–protein interactions, cationic arginine-rich peptides with a combination of various neuroprotective properties, shuttle peptides that ensure the permeability of neuroprotectors through the blood–brain barrier, and synthetic peptides that mimic natural regulatory peptides and hormones. In this review, we consider the latest achievements and trends in the development of new biologically active peptides, as well as the role of transcriptomic analysis in identifying the molecular mechanisms of action of potential drugs aimed at the treatment of ischemic stroke.

Background: Currently, the most dynamic areas in the glutamate receptor system neurobiology are the identification and development of positive allosteric modulators (PAMs) of glutamate ionotropic receptors. PAM-based drugs are of great interest as promising candidates for the treatment of neurological diseases, such as epilepsy, Alzheimer's disease, schizophrenia, etc. Understanding the molecular mechanisms underlying the biological action of natural and synthetic PAMs is a key point for modifying the original chemical compounds as well as for new drug design. Objective: We are trying to elaborate a system of molecular functional screening of ionotropic glutamate receptor probable PAMs. Methods: The system will be based on the radioligand - receptor method of analysis and will allow rapid quantification of new AMPAR probable PAMs molecular activity. We plan to use a tritiumlabeled analogue of recently elaborated ionotropic GluR probable PAM ([3H]PAM-43) as the main radioligand. Results: Here, we characterized the specific binding of the ligand and its ability to potentiate ionotropic GluR currents. The existence of at least two different sites of [3H]PAM-43 specific binding has been shown. One of the above sites is glutamate-dependent and is characterized by higher affinity. “Patchclamp” technique showed the ability of PAM-43 to potentiate ionotropic GluR currents in rat cerebellar Purkinje neurons in a concentration-dependent manner. Conclusion: The possibility of using PAM-43 as a model compound to study different allosteric effects of potential regulatory drugs (AMPAR allosteric regulators) was shown. [3H]PAM-43 based screening system will allow rapid selection of new AMPAR probable PAM structures and quantification of their molecular activity.

Neurodegenerative diseases and depression are multifactorial disorders with a complex and poorly understood physiopathology. Astrocytes play a key role in the functioning of neurons in norm and pathology. Stress is an important factor for the development of brain disorders. Here, we review data on the effects of stress on astrocyte function and evidence of the involvement of astrocyte dysfunction in depression and Alzheimer’s disease (AD). Stressful life events are an important risk factor for depression; meanwhile, depression is an important risk factor for AD. Clinical data indicate atrophic changes in the same areas of the brain, the hippocampus and prefrontal cortex (PFC), in both pathologies. These brain regions play a key role in regulating the stress response and are most vulnerable to the action of glucocorticoids. PFC astrocytes are critically involved in the development of depression. Stress alters astrocyte function and can result in pyroptotic death of not only neurons, but also astrocytes. BDNF-TrkB system not only plays a key role in depression and in normalizing the stress response, but also appears to be an important factor in the functioning of astrocytes. Astrocytes, being a target for stress and glucocorticoids, are a promising target for the treatment of stress-dependent depression and AD.

The phytohormone abscisic acid (ABA) plays an important role in plant growth and in response to abiotic stress factors. At the same time, its accumulation in soil can negatively affect seed germination, inhibit root growth and increase plant sensitivity to pathogens. ABA is an inert compound resistant to spontaneous hydrolysis and its biological transformation is scarcely understood. Recently, the strain Rhodococcus sp. P1Y was described as a rhizosphere bacterium assimilating ABA as a sole carbon source in batch culture and affecting ABA concentrations in plant roots. In this work, the intermediate product of ABA decomposition by this bacterium was isolated and purified by preparative HPLC techniques. Proof that this compound belongs to ABA derivatives was carried out by measuring the molar radioactivity of the conversion products of this phytohormone labeled with tritium. The chemical structure of this compound was determined by instrumental techniques including high-resolution mass spectrometry, NMR spectrometry, FTIR and UV spectroscopies. As a result, the metabolite was identified as (4RS)-4-hydroxy-3,5,5-trimethyl-4-[(E)-3-oxobut-1-enyl]cyclohex-2-en-1-one (dehydrovomifoliol). Based on the data obtained, it was concluded that the pathway of bacterial degradation and assimilation of ABA begins with a gradual shortening of the acyl part of the molecule.

The effects of the peptide ACTH(6-9)-Pro-Gly-Pro at doses of 5; 50; 500 μg/kg on the Wistar rats' behaviour and gut mucosal microbiota composition under conditions of chronic immobilization stress (CRS) were studied. CRS increased anxiety-like and depressive-like behaviour, disturbances in locomotor activity and gut dysbiosis. Administration of ACTH(6-9)-Pro-Gly-Pro showed many phenotypic results. Peptide demonstrated anti-depressant activity at doses of 5 and 500 μg/kg by a decrease in the total immobile time in the FST. ACTH(6-9)-Pro-Gly-Pro administered at a dose of 50 μg/kg resulted in an anxiolytic effect which is shown by an increase in the time in the open arms of EPM (p 0.05) and a decrease in the time in the closed arms (p 0.05). Moreover, the peptide led to a decrease in alpha- and beta-diversity of the gut microbiota (p 0.01). Correlation and linear regression analysis demonstrated central mechanisms of ACTH(6-9)-Pro-Gly-Pro anxiolytic activity and both central and peripheral ones in an anti-depressant effect. In this way, peptide ACTH(6-9)-Pro-Gly-Pro could prevent the development of behavioural disturbances and gut dysbiosis caused by chronic restraint stress.

The phytohormone abscisic acid (ABA) plays an important role in plant growth and in response to abiotic stress factors. At the same time, its accumulation in soil can negatively affect seed germination, inhibit root growth and increase plant sensitivity to pathogens. ABA is an inert compound resistant to spontaneous hydrolysis and its biological transformation is scarcely understood. Recently, the strain Rhodococcus sp. P1Y was described as a rhizosphere bacterium assimilating ABA as a sole carbon source in batch culture and affecting ABA concentrations in plant roots. In this work, the intermediate product of ABA decomposition by this bacterium was isolated and purified by preparative HPLC techniques. Proof that this compound belongs to ABA derivatives was carried out by measuring the molar radioactivity of the conversion products of this phytohormone labeled with tritium. The chemical structure of this compound was determined by instrumental techniques including high-resolution mass spectrometry, NMR spectrometry, FTIR and UV spectroscopies. As a result, the metabolite was identified as (4RS)-4-hydroxy-3,5,5-trimethyl-4-[(E)-3-oxobut-1-enyl]cyclohex-2-en-1-one (dehydrovomifoliol). Based on the data obtained, it was concluded that the pathway of bacterial degradation and assimilation of ABA begins with a gradual shortening of the acyl part of the molecule.

Brain stroke continues to claim the lives of million people every year. To build the effective strategies for stroke treatment it is necessary to understand the neuroprotective mechanisms that are able to prevent the ischemic injury. Consisting of the ACTH(4-7) fragment and the tripeptide Pro-Gly-Pro (PGP), the synthetic peptide Semax effectively protects brain against ischemic stroke. However, the molecular mechanisms underlying its neuroprotection and participation of PGP in them are still needed to be clarified. To reveal biological processes and signaling pathways, which are affected by Semax and PGP, we performed the transcriptome analysis of cerebral cortex of rats with focal cerebral ischemia treated by these peptides. The genome-wide biochip data analysis detected the differentially expressed genes (DEGs) and bioinformatic web-tool Ingenuity iReport found DEGs associations with several biological processes and signaling pathways. The immune response is the process most markedly affected by the peptide: Semax enhances antigen presentation signaling pathway, intensifies the effect of ischemia on the interferon signaling pathways and affects the processes for synthesizing immunoglobulins. Semax significantly increased expression of the gene encoding the immunoglobulin heavy chain, highly affects on cytokine, stress response and ribosomal protein-encoding genes after occlusion. PGP treatment of rats with ischemia attenuates the immune activity and suppresses neurotransmission in the CNS. We suppose that neuroprotective mechanism of Semax is realized via the neuroimmune crosstalk, and the new properties of PGP were found under ischemia. Our results provided the basis for further proteomic investigations in the field of searching Semax neuroprotection mechanism.

It was shown that the anxiolytic effect of Selank is comparable to that of classical benzodiazepine drugs and that the basis of their mechanism of action may be similar. These data suggest that the presence of Selank may change the action of classical benzodiazepine drugs. To test this hypothesis, we evaluated the anxiolytic activity of Selank and diazepam in rats both under conditions of unpredictable chronic mild stress and in its absence, after the individual and combined administration of these compounds using the elevated plus maze test. We found that, even in the absence of chronic stress, the administration of a course of test substances changed anxiety indicators toward their deterioration, but the changes after the administration of a course of Selank were less pronounced. In conditions of chronic stress, anxiety indicator values after the simultaneous use of diazepam and Selank did not differ from the respective values observed before chronic stress exposure. The data obtained indicate that the individual administration of Selank was the most effective in reducing elevated levels of anxiety, induced by the administration of a course of test substances, whereas the combination of diazepam with Selank was the most effective in reducing anxiety in unpredictable chronic mild stress conditions.

RATIONALE Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction. METHODS Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value. RESULTS After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected. CONCLUSIONS Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative analysis of the hypocholesterolemic and antithrombotic action of small regulatory glyproline peptides (Pro-Gly-Pro, Arg-Pro-Gly-Pro and Pro-Gly-Pro-Leu) was performed on an experimental hypercholesterolemia model of rats. Repeated intranasal introduction of glyproline peptides to fat-diet-fed animals led to more active functioning of the anticoagulation system (the anticoagulant and fibrinolytic properties of the plasma increased and platelet aggregation decreased) and to normalization of the total cholesterol level as a parameter of lipid metabolism. The largest anticoagulant and hypocholesterolemic effect was detected for the Pro-Gly-Pro-Leu peptide. Hypothetical mechanisms of antithrombotic and hypocholesterolemic effects of glyproline peptides are presented.

Parkinson’s disease (PD) is the second most common severe neurodegenerative disorder. It is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Unfortunately, PD remains incurable. Therapy based on regulatory peptides, particularly neuroprotective peptides, which can sustain or activate neuron plasticity to enable their survival and function in difficult environments and after violated homeostasis, is a promising approach to cure PD. Some studies show that the synthetic analogs of natural peptides may be used as an etiological or at least a complementary therapy in PD. Therefore, in the present pilot study, we investigated the effects of the synthetic peptides Semax and dopamine neuron stimulating peptide (DNSP-5), and a new synthetic Semax-DNSP-5 hybrid peptide (SD) on the functioning of brain neurons. An analysis of the levels of dopamine (DA), noradrenaline (NA), 5-hydroxytriptamine (5-HT), an expression analysis of Gdnf and Gdnf receptor genes Gfra1, Gfra2, Gfra3, Gfra4, and Gfral in various regions of the brain of rats with 6-OHDA-induced PD-like parkinsonism, and a study of the motor activity of the rats in an “open field” test showed that DNSP-5 and SD elevated the level of DA in the nonlesioned striatum. DNSP-5 also increased the expression of Gfra1 and Gfra2 in the nonlesioned striatum and lesioned substantia nigra (SN) which suggested that DNSP-5 had compensatory and neuroprotective properties. SD demonstrated similar, albeit less pronounced effects to DNSP-5 on DA metabolism and gene expression. Of the peptides studied, only SD tended to increase the horizontal and vertical activity of rats. In conclusion, these findings suggest that DNSP-5 and SD have potential neuroprotective properties and may stimulate the surviving DA neurons.

The peptide Semax effectively protects brain tissues against ischemic stroke. However, the molecular mechanisms that underlie its action remain unknown. We used the focal cerebral ischemia rat model with permanent middle cerebral artery occlusion (pMCAO). During the experiments, animals were given intraperitoneal injections of Semax, Pro-Gly-Pro, or saline. We studied the effect of the peptides on the expression of more than 80 growth factor genes in the cortex. As a response to Semax administration, alterations in the expression of growth factor genes were detected at 3, 24, and 72 h after pMCAO. The most pronounced effects of Semax, i.e., the downregulation of the transcripts of 20 genes and the upregulation of 12 growth factor genes, were observed 3 days after artery occlusion. According to our data, Semax promoted the upregulation (by ≥10-fold) of the Csf3 and Artn genes, as well as of the cytokine genes Il1b and Il6. The peptide products of these genes have regulatory properties and exert neuroprotective effects in injured brain tissues. We presume that Semax triggers neuroprotective mechanisms by affecting these systems via the regulation of the expression of growth factor genes.

The effect of synthetic nucleoside derivatives on the growth and survival of cultured human ovarian cancer cells (line SKOV-3) and normal human lung fibroblasts has been investigated. The dialdehyde derivatives of uridine, 1-β-D-erythrofuranosyl uracil and 3′-O-β-D-ribofuranosyl-2′-deoxythymidine exhibited a marked toxic effect on SKOV-3 cells, while their unoxidized analogues did not exhibit such action. Cultured human fibroblasts were less susceptible to the damaging effect of the dialdehyde nucleosides. The dialdehyde derivative of 1-β-D-erythrofuranosyl uracil demonstrated the highest difference in its cytotoxic effect on these cultures: 50% or higher suppression of SKOV-3 cells growth was achieved at the concentrations of this compound ten times lower than in the case of normal fibroblasts.

We describe here our studies of the effects of single doses and courses of the synthetic tuftsin analog Selank (a peptide with the sequence Thr-Lys-Pro-Arg-Pro-Gly-Pro) at a dose of 200 μg/kg on the transcription profile of rat hippocampal cells using a cDNA microarray method. Single doses of Selank produced more than two-fold changes in the mRNA levels of 36 genes, while courses were followed by changes in 20 genes. Most of these genes encoded proteins associated with the plasma membrane (including transmembrane proteins), suggesting that Selank is able to regulate ion homeostasis of hippocampal cells, thus modulating the levels of various ion-dependent processes at the molecular genetic level, these including learning and memory formation processes.

Tripeptide Pro-Gly-Pro (PGP) belongs to the group of biologically active regulatory peptides that are resistant to peptidases. This makes it possible to use it as a C-terminal fragment of peptide agents Semax and Selank. In recent years, the independent effects of PGP were revealed. The question was raised of whether PGP contributes to the action of pharmaceutical agents that contain this peptide as a fragment. Genome-wide analysis was performed to examine the PGP impacts on the transcriptome of rat cerebral cortex in experimental ischemia. We compared the gene expression levels in animals exposed to ischemia and PGP and in rats of the ischemia control group in 3 and 24 h after the permanent occlusion of the left middle cerebral artery. The altered expression of 29 genes was detected in 3 h, while the altered expression of 57 genes was detected in 24 h. The proteins encoded by these genes fulfill various functions, such as cytokines, transport proteins, transcription factors, transmembrane receptors, etc. Biological processes associated with the genes with altered expression under experimental conditions were determined. It was shown that PGP affects a number of processes involved in the functioning of various body systems. The expression of genes associated with the immune response process changed most significantly a day after the occlusion of the middle cerebral artery. The predominant effect of the suppression of the expression of the immune system genes was observed.

Solid-state catalytic isotope exchange of hydrogen under the action of spillover hydrogen in organic compounds applied on a nonorganic carrier has been studied. The deuterium-labeled peptide [D]dalargin containing 14 deuterium atoms, [D]melatonin, and [D]histamine with the 72–92% substitution degree of all C–H bonds were prepared by this reaction. The activation energy of hydrogen isotope exchange with T2 and D2 in glycine and α-aminoisobutyric acid was obtained experimentally. It was shown that for the studied reaction the kinetic isotopic effect is 1.2–1.4 by the Hartree–Fock method, which is several times smaller than one in the liquid-phase reactions. Quantum chemical calculations of the isotope shift values in the electronic spectra of deuterium-labeled metal ion complexes were performed using the RB3LYP/LanL2DZ and CIS/LanL2DZ methods for calculation of the ground and the excited states. It was shown for deuterium-labeled histamine complexes [D12]Pd(him)2Cl2 and [D12]Cu(him)2Cl that the isotopic eff...

Natural regulatory peptides are biologically active compounds that are produced by various cells and provide a link among the main regulatory systems of the body. The field of research into the biologic activity of endogenous regulatory peptides is extremely vast. These peptides affect the cardiovascular, immune, reproductive, endocrine, digestive, and other systems, alter energy metabolism, and are especially effective in the regulation of the central nervous system. Despite of the wide range of preventive and therapeutic effects of natural regulatory peptides and proteins, their application in clinical practice is difficult. This is primarily because of their extreme instability, as they are rapidly degraded by proteases of the gastrointestinal tract, blood, cerebrospinal fluid, and other biologic media. Compounds with higher stability (i.e., a considerably longer half-life compared with that of natural molecules) and the ability to provide a directional effect on the various body systems were obtained from modifications of endogenous regulatory peptides. Synthetic analogs of regulatory peptides, as a rule, contain only natural amino acids in their composition, and their biodegradation does not lead to the formation of toxic products; thus, they have fewer side effects. This review focuses on the consideration of two synthetic regulatory peptides, Semax and Selank, which were the bases for the creation of new drugs that are used effectively in the treatment of various diseases of the nervous system. The synthetic analog of an adrenocorticotropic hormone 4-10 fragment (ACTH4-10) Semax is a powerful neuroprotective agent that is particularly effective as a therapy for stroke. Selank was synthesized on the basis of the natural immunomodulator tuftsin. Selank is a powerful anxiolytic that is used as a therapy for generalized anxiety disorder and neurasthenia without sedative and muscle-relaxant effects. This review presents the results of research aimed at studying the influence of these peptides on the transcriptome of brain cells. The problems of drugs developed based on the clinical activities of Semax and Selank are discussed separately.

Brain stroke continues to claim the lives of million people every year. To build the effective strategies for stroke treatment it is necessary to understand the neuroprotective mechanisms that are able to prevent the ischemic injury. Consisting of the ACTH

Clinical studies have shown that Selank had an anxiolytic effect comparable to that of classical benzodiazepine drugs, which can enhance the inhibitory effect of GABA by allosteric modulation of GABAA receptors. These data suggest that the molecular mechanism of the effect of Selank may also be related to its ability to affect the performance of the GABAergic system. To test this hypothesis, we studied the changes in expression of 84 genes involved in the functioning of the GABAergic system and in the processes of neurotransmission in the culture of neuroblastoma IMR-32 cells using qPCR method. As test substances, in addition to Selank, we selected the major GABAA receptor ligand, GABA, the atypical antipsychotic, olanzapine, and combinations of these compounds (Selank and GABA; Selank and olanzapine). We found no changes in the mRNA levels of the genes studied under the effect of Selank. The combined effect of GABA and Selank led to nearly complete suppression of changes in expression of genes in which mRNA levels changed under the effect of GABA. When Selank was used in conjunction with olanzapine, the expression alterations of more genes were observed compared with olanzapine alone. The data obtained indicate that Selank has no direct effect on the mRNA levels of the GABAergic system genes in neuroblastoma IMR-32 cells. At the same time, our results partially confirm the hypothesis that the peptide may affect the interaction of GABA with GABAA receptors. Our data also suggest that Selank may enhance the effect of olanzapine on the expression of the genes studied.

Clinical studies have shown the similarity of the spectrum of physiological effects of Selank and classical benzodiazepines, such as diazepam and phenazepam. These data suggest that there is a similar basis of their mechanism of action. To test this hypothesis we studied the effect of Selank and GABA on the expression of genes involved in the neuroreсeption and neurotransmission. We analyzed the expression of 84 genes involved in neuroreсeption (e.g., major subunit of the GABA receptor, transporters, ion channels, dopamine and serotonin receptors) in the frontal cortex of rats 1 and 3 hours after the administration of Selank or GABA (300 μg/kg) using real-time PCR method. We found significant changes in the expression of 45 genes 1 hour after the administration of the compounds. Three hours after Selank or GABA administration, 22 genes changed their expression. We found positive correlation between the changes in genes expression within 1 hour after administration of Selank or GABA. Our results showed that Selank caused a number of alterations in the expression of genes involved in neuroreсeption. The data obtained indicate that Selank is characterized by its complex effects on nerve cells, and one of its possible molecular mechanisms is associated with allosteric modulation of the GABAergic system.

Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction.Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value.After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected.Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright © 2016 John WileySons, Ltd.

Neurotrophins regulate key functions of nervous tissue cells. Analysis of neurotrophin mRNA expression is an appropriate tool to assess therapeutic efficiency of antistroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide on mRNA expression of neurotrophins Ngf, Bdnf, and Nt-3 and their receptors TrkA, TrkB, TrkC, and p75 in rat frontal cortex, hippocampus, and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated at 30 min and 1, 2, 4, 8, 12, and 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our results demonstrated that ischemia caused a significant decrease in gene expression in the hippocampus. Semax and PGP treatment affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of neurotrophin receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides was observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the mechanism of Semax action and reveal certain features of mRNA expression of neurotrophins and their receptors under experimental conditions.

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.

Modern theories about the processes that take place inside solvent-free catalyst systems are considered and the various factors that have an effect on the successful introduction of tritium into organic compounds are summarized. Obtaining labelled compounds using solvent-free catalyst systems, liquid-phase methods, and isotopic exchange with tritiated water are all regarded as various manifestations of the same set of occurrences in the presence of tritium, a substrate and a catalyst. The use of various isotopic exchange methods has made possible the introduction of tritium into practically any type of biologically active compound. This will enable scientists to carry out more detailed studies of the processes inside living organisms. Copyright © 2010 John Wiley & Sons, Ltd.

Photochemically induced thrombosis of blood vessels in the prefrontal cortex in rats was shown to lead to ischemic infarcts in the lesion zone. Bilateral ischemic lesioning of the prefrontal cortex degraded measures of spatial memory when animals were tested in a Morris water maze with an invisible platform 20-24 days after surgery. Chronic intranasal administration of the peptide Met-Glu-His-Phe-Pro-Gly-Pro (Semax), a synthetic analog of ACTH(4-7), at a dose of 250 microg/kg/day during the first six days after photothrombosis, led to recovery of the animals' learning ability. The long-term antiamnestic action of the peptide observed here may result from its neuroprotective activity and its ability to stimulate the synthesis of neurotrophic factors.

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation, and maintenance of function of different neuron populations. Some peptides are able to affect the production and activity of neurotrophins. One of these synthetic peptides is heptapeptide Semax, an analog of the N-terminal adrenocorticotropic hormone fragment 4-10. It is known that Semax has effects on learning and memory formation and exerts some neuroprotective effects in rodents and humans. Male Wistar rats were treated for 20 min, 40 min, 90 min, 3 h, 8 h, and 24 h with Semax. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) gene expression in rat brain and retina was analyzed by real-time polymerase chain reaction. It was revealed that after Semax administration the multidirectional activation of the expression of the genes under investigation in the hippocampus, frontal cortex, and retina was observed. The expression of both neurotrophin genes was decreased in rat hippocampus and retina 20 min after Semax administration and was increased in the frontal cortex. The expression levels of NGF remained practically constant in the retina at the initial stage, whereas the expression levels of BDNF were significantly increased 90 min after Semax administration.

Semax is a synthetic peptide consisting of the ACTH fragment (4–7) and the C-terminal Pro-Gly-Pro peptide. It promotes neuron survival during hypoxia and increases selcective attention and memory consolidation. It has been shown that semax influences the expression of some genes, particularly nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). In the present study, we performed an analysis of the time course of the expression of NGF and BDNF genes in the hippocampus and frontal cortex in rats after a single intranasal administration of semax at a dose of 50 μg/kg. We found that semax administration resulted in a rapid long-term activation of the NGF and BDNF genes expression, which was specific for different rat brain structures investigated.

We describe a method of preparation of high specific activity tritium-labelled leukotriene (LT) B4 from [5,6,8,9,11,12,14,15-3H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB4-synthesizing enzyme system from rat basophilic leukemia (RBL-1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 5′-triphosphate induced [3H] AA transformation to [3H] LTB4. In optimized conditions up to 15% of total radioactivity of the incubation mixture was present in [3H] LTB4. A separation of [3H] LTB4 from other labelled C20:4 products was achieved by a three-step reverse phase-high-performance liquid chromatography in methanol- and acetonitrile-based solvent systems. [3H] LTB4 was confirmed to be identical to the naturally occurring LTB4 by a radioligand binding assay using a culture of HF1 cells that express a BLT1 receptor. Copyright © 2008 John Wiley & Sons, Ltd.

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 1 h with Semax (50 μg/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.

The solid-phase method for hydrogenolysis, hydrogenation and isotope exchange reactions is described. Its potential for tritium labelling of organic compounds including lipids, nucleotides, amino acids, peptides and pharmaceuticals is demonstrated. The influence of the reaction conditions on the yield and specific radioactivity of the labelled compounds is considered. It is shown that results can be interpreted by the dependence of tritium spillover reactions on the nature of the initial compound and other factors. Copyright © 2007 John Wiley & Sons, Ltd.

Semax had a pronounced neuroprotective and antiamnesic effect during focal photoinduced ischemia of the prefrontal cortex. Intranasal administration of Semax for 6 days decreased the volume of cortical infarction and improved retention and performance of conditioned passive avoidance response.

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 μg/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.

Isotopic effect of electron excitation in organic compounds was for the first time experimentally and theoretically analyzed. The nature of this phenomenon is due to zero-point energies (ZPE) for the ground and the excited states of the isotope substituted tryptophan. l -[ 3 H]tryptophan ( l -[ 3 H]Trp) with a tritium–hydrogen substitution ratio of 0.2–0.45 has been produced by high-temperature solid-state catalytic isotope exchange and was used in an experimental study. A comparison of the UV spectra of l -[ 3 H]Trp and l -[ 1 H]Trp showed a short-wavelength shift of the absorption maximum in the tritium-substituted compound that corresponds to an energy of 130 cal/mol for the electronic transition. To account for this effect, quantum chemical calculations of the geometric and electronic structures, frequencies of normal vibrations, and transition energies have been performed using of the restricted Hartree–Fock method (RHF), density-functional theory (RB3LYP), and configuration interaction (RCIS). The shifts of all allowed electronic transitions have been shown to shift towards the short-wavelength region and to be equal to 152 cal/mol for the first singlet–singlet transition (within the 280 nm region), to 242 cal/mol for the third transition (within the 220 nm region), and to 557 cal/mol for the singlet–triplet transition. All the shifts obtained by calculations are due to zero-point energies for the ground and the excited states of [ 3 H]Trp and [ 1 H]Trp.

Various mechanisms of hydrogen spillover are discussed. A number of evidences are given that tritium incorporation by isotope exchange occurs with the participation of both tritium cations and atomic tritium. The molar radioactivity of the labeled products prepared at temperatures of up to 180°C is, apparently, mainly determined by reactions of the organic compound with tritium cations, and the degree of isotope exchange depends chiefly on the efficiency of tritium spillover to the bulk of the organic substance supported on the catalyst.

The proliferation, differentiation and survival of neuronal and glial cells are affected by a number of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and others. In a previous study, we observed the effects of ‘Semax’ (Met-Glu-His-Phe-Pro-Gly-Pro), the physiologically active analogue of adrenocorticotropic hormone(4–10), on neuronal cell survival in vitro. We hypothesized that these effects may be mediated by the regulation of expression of some neurotrophic factors. To test this hypothesis we analyzed NGF and BDNF gene expression in glial cells obtained from the basal forebrain of newborn rats, following in vitro treatment with ‘Semax’. We observed changes in mRNA levels for both the NGF and BDNF genes. The greatest increase in expression was found after 30 min of ‘Semax’ administration. At this time, BDNF mRNA level was increased eight-fold in comparison with control, and NGF mRNA level was increased five-fold.

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.

Previous studies have shown that synthetic tuftsin analogue Selank and its fragments cause a number of alterations in the expression of certain genes involved in inflammation in mouse spleen. In this work we studied the effect of Selank and its short fragment Gly-Pro on the temporary dynamics of C3, Casp1, Il2rg, and Xcr1 genes expression in mouse spleen after single intraperitoneal injection (100 μg/kg) of peptides using real-time PCR method. We found a significant 3-fold decrease in the C3 mRNA level just 30 min after Selank injection and similar alteration this gene mRNA level after Gly-Pro administration. A wave-like alteration in the Casp1 mRNA level was observed after Selank injection. We found a significant alteration in the mRNA level of the Il2rg gene at early time points after Selank and Gly-Pro administration and an almost equal reduction in the Xcr1 mRNA level 90 min after the administration of Selank and its fragment. Our results showed that, Selank and its short fragment Gly-Pro influence the expression of genes that mediate different types of immune responses, thereby maintaining the balance of the immune system. It should be noted that in most cases, there was a coincidence in the expression profiles of the studied genes after Selank and Gly-Pro administration. This might indicate an active contribution of the dipeptide to the final effect of Selank.

The synthetic peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is used successfully in acute stroke therapy. In spite of numerous studies on the subject, many aspects of the neuroprotective effects of the peptide remain unknown. We studied the action of Semax and its C-terminal tripeptide Pro-Gly-Pro on the expression of the VEGF gene family (Vegf-a, Vegf-b, Vegf-c, Vegf-d, and Plgf) and their receptors (Vegfr-1, Vegfr-2, and Vegfr-3) in the frontoparietal cortex region of the rat brain at 3, 24, and 72 h after permanent left middle cerebral artery occlusion (pMCAO). The relative mRNA level of the genes studied was assessed using real-time reverse transcription-PCR. The Vegf-b and Vegf-d genes were most affected by the peptides, which resulted in their most noticeable activation at 3 h after pMCAO. The level of Vegf-d transcripts decreased considerably, whereas the mRNA level of the Vegf-b gene was significantly increased after 72 h of treatment with each of the peptides. In addition, the effects of the peptides on the expression of the Vegf-b and Vegf-d genes were the opposite of the action of ischemia. It is suggested that the identified effects of the peptides diminish the effects of ischemia, thus participating in the positive therapeutic effect of Semax on ischemic stroke.