Water-soluble noncovalent adducts of the heterometallic copper subgroup complexes and human serum albumin with remarkable luminescent properties.

Novel water-soluble noncovalent adducts of the heterometallic copper subgroup complexes and human serum albumin (HSA) display strong phosphorescence, internalize into HeLa cells and can be used in time-resolved fluorescent imaging.

[Synthesis of GnRH analogs and their application in targeted gene delivery systems].

A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.

[Amides of creatine: perspectives of neuroprotection].

We evaluated the efficacy of derivatives of creatine and amino acids (CrAA) for decreasing cerebral injury in rats with transient middle cerebral artery occlusion (MCAO). Neuroprotective effects of amides of creatine and glycine (CrGlyOEt), phenylalanine (CrPheNH2), thyrosine (CrTyrNH2), and GABA (CrGABAOEt) were investigated. Brain injury was evaluated on day 2 after transient MCAO using a TTC staining of brain slices. Compared with the MCAO control group, all the CrAms showed decreased cerebral injury (p < 0.05). However CrPheNH2, CrTyrNH2, and CrGABAOEt were toxic after intravenous administration and investigated only after intraperitoneal injection. CrGlyOEt did not show any toxicity at dose of 1 mmol/kg. These data evidenced that creatinyl amides can represent promising candidates for the development of new drugs useful in brain ischemia treatment.

[GnRH analogues containing SV-40 virus T-antigen nuclear localization sequence].

To improve the efficiency of anticancer drugs due to their delivery to intracellular targets a set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized. NLS was attached to the parent molecule via ε-amino group of D-Lysine in position 1 or 6 of peptide sequence using orthogonal protection strategy. The biological activity studies revealed that incorporation of NLS moiety significantly increases cytotoxic activity of palmitoyl-containing GnRH analogues in vitro. The influence of tested peptides on tumor cells does not accompanied by the destruction of cell membrane, as confirmed in experiments with normal fibroblasts, used as a control.

[Non-viral gene therapy approach for regenerative recovery of skin wounds in mammals].

The rate and character of skin tissue regeneration after wounds, burns and other traumas depend on the cell proliferation within damaged area. Acceleration of healing by stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment consisting in the transfer of genes encoding mitogenic growth factors to wound area. The most important step in the development of gene therapy approaches is the design of gene delivery tools. In spite of high efficacy of viral vectors, the non-viral means have some preferences (low toxicity, low immunogenity, safety and the absence of backside effects). Among non-viral gene delivery tools, molecular conjugates are the most popular because of their efficacy, simplicity, and the capacity to the targeted gene transfer. In the present work we have developed two molecular conjugates--NLS-TSF7 and NLS-TSF12 consisting of the modified signal of nuclear localization of T-antigen of SV40 virus (cationic part) and the peptide ligands of mammalian transferrin receptor (ligand part). These conjugates bind to plasmid DNA with formation of polyelectrolytic complexes and are capable to deliver plasmid DNA into cells expressing transferrin receptors by receptor-mediated endocytosis. Transfer of the expression vector of luciferase gene in the complex with molecular conjugate NLS-TSF7 to murine surface tissues led to about 100 fold increasing of luciferase activity in comparison with the transfer of free expression vector. Treatment of slash wounds in mice with the complexes of expression vector of synthetic human gene encoding insulin-like growth factor 1 with molecular conjugates NLS-TSF7 led to acceleration of healing in comparison with mice treated with free expression vector. The results obtained confirm the high efficiency of the developed regenerative gene therapy approach for the treatment of damaged skin tissues in mammals.

[Transfer of genetic constructions through the transplacental barrier into mice embryos].

Genetic modification of mammalian embryos is an important way to model various changes in human development; also, it is an instrument for studying the functions of certain genes in mammals. Using our own experience in developing modes of delivery of genetic constructions to mammals in a nonviral way, we present here data on the delivery of a eukaryotic expression vector to mice embryos through the transplacental barrier with the use of hydrodynamic intravenous injections of DNA-hybrid peptide complexes to pregnant females. The peptide has a cationic part for interaction with DNA and includes a ligand structure towards receptors of the releasing factor of luteinizing hormone (RFLH, luliberin). Advantages of the suggested method are simplicity, economy, nonimmunogenicity for females, and the ability to multiply repeat the procedure. On the basis of the method, systemic gene delivery into tissues of mammalian embryos may be developed.

Aggregation work at polydisperse micellization: ideal solution and "dressed micelle" models comparing to molecular dynamics simulations.

General thermodynamic relations for the work of polydisperse micelle formation in the model of ideal solution of molecular aggregates in nonionic surfactant solution and the model of "dressed micelles" in ionic solution have been considered. In particular, the dependence of the aggregation work on the total concentration of nonionic surfactant has been analyzed. The analogous dependence for the work of formation of ionic aggregates has been examined with regard to existence of two variables of a state of an ionic aggregate, the aggregation numbers of surface active ions and counterions. To verify the thermodynamic models, the molecular dynamics simulations of micellization in nonionic and ionic surfactant solutions at two total surfactant concentrations have been performed. It was shown that for nonionic surfactants, even at relatively high total surfactant concentrations, the shape and behavior of the work of polydisperse micelle formation found within the model of the ideal solution at different total surfactant concentrations agrees fairly well with the numerical experiment. For ionic surfactant solutions, the numerical results indicate a strong screening of ionic aggregates by the bound counterions. This fact as well as independence of the coefficient in the law of mass action for ionic aggregates on total surfactant concentration and predictable behavior of the "waterfall" lines of surfaces of the aggregation work upholds the model of "dressed" ionic aggregates.

In vivo neuroprotection by a creatine-derived compound: phosphocreatine-Mg-complex acetate.

Phosphocreatine-Mg-complex acetate (PCr-Mg-CPLX) is a creatine-derived compound that in previous in vitro research was able to increase neuronal creatine independently of the creatine transporter, thus providing hope to cure the hereditary syndrome of creatine transporter deficiency. In previous research we showed that it reproduces in vitro the known neuroprotective effect of creatine against anoxic damage. In the present paper we investigated if PCr-Mg-CPLX reproduces this neuroprotective effect in vivo, too. We used a mouse model of transient middle cerebral artery occlusion. Mice received PCr-Mg-CPLX or a mixture of the two separate compounds phosphocreatine (PCr) and MgSO(4), or vehicle. The injections were done 60 min and 30 min before ischemia. Forty-eight hours after ischemia neurological damage was evaluated with Clark's behavioural tests, then the infarct volume was measured. PCr-Mg-CPLX reduced the infarct volume by 48%, an effect that was not duplicated by the separate administration of PCr and MgSO(4) and the neurological damage was decreased in a statistically significant way. We conclude that PCr-Mg-CPLX affords in vivo neuroprotection when administered before ischemia. These results are comparable to previous research on creatine administration in experimental stroke. PCr-Mg-CPLX maintains creatine-like neuroprotective effects in vivo as well as in vitro. Our study suggests that PCr-Mg-CPLX might have a therapeutic role in the treatment of hereditary creatine transporter deficiency and of conditions where there is a high risk of impending stroke or cerebral ischemic damage, like high-risk transient ischemic attacks, open heart surgery, and carotid surgery.

[Derivatives of N-amidinoproline and their use in conventional and solid phase peptide synthesis].

N-Amidinoproline, a hybrid structure modeling key features of the Arg-Pro sequence, was synthesized. The activation of carboxyl group of free N-amidinoproline was found to result in the formation of a cyclic side product, whose structure was confirmed by ESI MS, 1H NMR, and 13C NMR spectra. The preparation of N-(mesitylenesulfonylamidino)-L-proline using the mesitylenesulfonyl derivative of 2-methylisourea was demonstrated to be accompanied by partial racemization. The target product was synthesized by modification of N-amidinoproline by mesitylenesulfonyl chloride. The possibility of using N-amidinoproline in the N-terminal modification of a peptide chain was shown by the example of synthesis of an analogue of the 95-98 fragment of fibrinogen alpha chain.

[Luliberin analogues exhibiting a cytotoxic effect on tumor cells in vitro].

Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized.

The creatine transporter mediates the uptake of creatine by brain tissue, but not the uptake of two creatine-derived compounds.

Hereditary creatine transporter deficiency causes brain damage, despite the brain having the enzymes to synthesize creatine. Such damage occurring despite an endogenous synthesis is not easily explained. This condition is incurable, because creatine may not be delivered to the brain without its transporter. Creatine-derived compounds that crossed the blood-brain barrier in a transporter-independent fashion would be useful in the therapy of hereditary creatine transporter deficiency, and possibly also in neuroprotection against brain anoxia or ischemia. We tested the double hypothesis that: (1) the creatine carrier is needed to make creatine cross the plasma membrane of brain cells and (2) creatine-derived molecules may cross this plasma membrane independently of the creatine carrier. In in vitro mouse hippocampal slices, incubation with creatine increased creatine and phosphocreatine content of the tissue. Inhibition of the creatine transporter with 3-guanidinopropionic acid (GPA) dose-dependently prevented this increase. Incubation with creatine benzyl ester (CrOBzl) or phosphocreatine-Mg-complex acetate (PCr-Mg-CPLX) increased tissue creatine content, not phosphocreatine. This increase was not prevented by GPA. Thus, the creatine transporter is required for creatine uptake through the plasma membrane. Since there is a strong indication that creatine in the brain is mainly synthesized by glial cells and transferred to neurons, this might explain why hereditary transporter deficiency is attended by severe brain damage despite the possibility of an endogenous synthesis. CrOBzl and PCr-Mg-CPLX cross the plasma membrane in a transporter-independent way, and might be useful in the therapy of hereditary creatine transporter deficiency. They may also prove useful in the therapy of brain anoxia or ischemia.

Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene.

[Delivery of "suicide" thymidine kinase gene of herpes virus in the complex with cationic peptide into human hepatoma cells in vitro]. / Dostavka "suitsidnogo" gena timidinkinazy virusa gerpesa v komplekse s kationnym peptidom v kletki gepatomy cheloveka in vitro.

The delivery of "suicide" herpes simplex virus type-1 thymidine kinase gene (tk) into tumor cells, followed by treatment with synthetic nucleotide analogues (gancyclovir, acyclovir), is a perspective approach to cancer therapy. Serious limitations in employment of the existing means of gene delivery into target cells constitute the main obstacle for cancer gene therapy development. In the present work a possibility to use a nonviral gene delivery system is shown based on the employment of lysine rich peptide K8 and amphipathic peptide JTS-1 for transferring tk gene into human hepatoma HepG2 cells. Cationic peptide K8 forms compact complexes with plasmid DNA, and JTS-1 acts as a pH-dependent endosomal releasing agent. Transfection of HepG2 cells by tk expression vector coupled with K8/JTS-1 peptides, followed by acyclovir administration (50-100 micrograms/ml) for 24 h leads to cell cycle arrest in the G1/S checkpoint of some cells, which eventually die through apoptosis. Treatment of HepG2 cells with higher acyclovir concentration (200 micrograms/ml) additionally results in a nonspecific toxic effect. The above results demonstrate the efficacy of K8/JTS-1 delivery system for the "suicide" cancer gene therapy, and may be regarded as a basis for further elaboration of "suicide" cancer approaches in vivo.

[Vasoactive properties of various fragments of human alpha-calcitonin gene-related peptide]. / Vazoaktivnye svoistva nekotorykh fragmentov al'fa-kal'tsitonin gen-rodstvennogo peptida cheloveka.

Effects of some fragments of alpha-calcitonine gene-related peptide (alpha-CGRP) on the collector lymphatic vessels of the rat small intestine mesenterium, were studied. All the fragments excepting alpha-CGRP (21-24) and (21-31) activate motility of the lymphatic vessels enhancing phasic contractions of the walls. Presence of aminoacid residuals in the 20-29 area is "critical" for realising vasoactive properties by these fragments.

Dynorphin A(6-12) analogs suppress thermal edema.

Dynorphin A (Dyn A) is a 17-residue opioid peptide derived from prodynorphin precursors found in mammalian tissues. Removal of Tyr1 from Dyn A produces a peptide that is more potent than Dyn A in attenuating the acute phase of the inflammatory response, as measured by inhibition of heat-induced edema in the anesthetized rat's paw (exposure to 58 degrees C water for 1 min). Dyn A(2-17), however, no longer interacts with opioid receptors. It was postulated that the non-opioid anti-inflammatory actions of Dyn A(2-17) may reside in Dyn A(6-12); that is, Arg-Arg-Ile-Arg-Pro-Lys-Leu. here we report on the activities of Dyn A(6-12) analogs modified by substitutions on the N terminus, by single N-methyl substitution and by single replacement of residues by alanine. The results indicated that the minimal sequence required for an anti-edema ED50 of <1.0 micromol/kg i.v. was anisoyl-Arg6-Arg7-Xaa8-Arg9-Pro10)-Xaa11-+ ++Xaa12-NH2. A prototype, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, with an ED50 of 0.20 micromol/kg i.v. compared to an ED50 of 0.08 micromol/kg i.v. for Dyn A(2-17), was selected for further tests of biological activity. This analog, like Dyn A(2-17), lowered blood pressure in anesthetized rats. In a model of neurogenic inflammation, produced by antidromic stimulation of the vagus in the anesthetized rat, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, 0.23 micromol/kg i.v., attenuated the negativity of tracheal tissue interstitial pressure (Pif), which normally develops after nerve stimulation. Modulation of interstitial pressure may be the mechanistic basis for the anti-edema properties of these Dyn A(6-12) analogs.

The presence of autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptor in the blood serum of patients with epilepsy.

We have analyzed the serum from patients with refractory epilepsy for the presence of autoreactive antibodies to AMPA glutamate receptor subunits. The presence and the level of autoantibodies were assessed using immunoblot and ELISA with synthetic peptides specific for subregions of AMPA glutamate receptor subunits. Patients with refractory epilepsy exhibited strong immunoreactivity to GluR1 subunit compared to healthy donors and patients with Parkinson's disease and Alzheimer's disease. Weak immunoreactivity to other AMPA glutamate receptor subunits was also detected and the signal was diminished in the raw GluR4>GluR3>GluR2. The occurrence of autoantibodies to specific neurotransmitter subunits in the sera of patients with refractory epilepsy suggest that autoimmune process may underlie this disorder.

Antithrombotic and vasorelaxant properties of a novel synthetic RGD peptide containing nitric oxide.

We have developed a novel synthetic peptide containing both the antiadhesive Arg-Gly-Asp (RGD) amino acid sequence and a nitric oxide (NO) moiety well known for its vasorelaxant properties. The main objective of this study was to determine whether this hybrid molecule is concurrently effective with regard to antithrombotic and vasorelaxation actions. Studies of in vitro platelet adhesion and of in vivo platelet thrombus formation in the rat demonstrated that the RGD-NO peptide increased the antithrombotic characteristics of the RGD peptide alone. The RGD-NO peptide also caused relaxation of rat aortic rings, while the RGD peptide did not induce relaxation. These characteristics of Ac-RGDC-SNO suggest that this or similar compounds may have potential as effective antithrombotic agents in coronary and peripheral artery disorders.

[The role of calcitonin gene-related peptide fragments in regulating the microcirculation in rats]. / O roli fragmentov kal'tsitonin-gen-rodstvennogo peptida v reguliatsii mikrotsirkuliatsii u krys.

Fragments of the calcitonin gene-related neuropeptide's molecule with the amino acid sequence 1-9, 10-20, 15-24, 20-29 and 30-37, were studied. The fragment 20-29 revealed the greatest biological activity: it induced a dose-dependent drop of arterial pressure in i.v. administration, enlarges arterial and venous microvessels when locally applied.

The action of LH-releasing hormone and five analogues on oestradiol, oxytocin and vasopressin secretion by bovine granulosa cells in culture.

The release of oxytocin, arginine-8-vasopressin and oestradiol-17 beta by bovine granulosa cells in culture was analysed either with or without LH-releasing hormone (LHRH), its agonists (cyclo [Pro1DPhe6] LHRH and des 1-3, 10[DAla6] LHRH) or antagonists ([DPhe2DPhe6] LHRH, [DPhe2DPhe(NH2)6] LHRH or cyclo [Pro1DPhe2DPhe6] LHRH). All preparations used stimulated granulosa oxytocin and oestradiol secretion. Vasopressin release was significantly increased after all treatments with LHRH antagonists, but not after LHRH or its agonists. Our observations demonstrated a direct influence of LHRH and its analogues on the secretion of oestrogen and nonapeptide hormones by bovine granulosa cells. A comparison of the effects of LHRH, its agonists and antagonists suggests that the action of these peptides at the hypophysial and ovarian level is relatively independent.