Melatonin and its bioisosteres as potential therapeutic agents for the treatment of retinopathy of prematurity.

We conducted a study on the impact of intraperitoneal injections of melatonin and its three bioisosteres (compounds 1-3) on the development of oxygen-induced retinopathy in newborn rats during a 21-day experiment. It was demonstrated that melatonin and its analogues 1-3 effectively reduce the total protein concentration in the vitreous body of rat pups, decrease concentration of VEGF-A, and lower the level of oxidative stress (as indicated by normalization of antioxidant activity in the vitreous body). Melatonin and its analogues 1-3 equally normalize the level of VEGF-A. Analogues 1 and 2 even exceed melatonin in their ability to reduce protein influx into the vitreous body. However, analogue 2 had no effect on antioxidant activity, while analogues 1 and 3 caused a significant increase in this parameter, with analogue 3 even slightly exceeding melatonin. Thus, it can be concluded that analogues 1-3 are comparable to melatonin and can be utilized as potential therapeutic agents for the treatment of retinopathy of prematurity.

A high-power synthesized ultrawideband radiation source.

A high-power ultrawideband radiation source has been developed which is capable of synthesizing electromagnetic pulses with different frequency bands in free space. To this end, a new circuit design comprising a four-channel former of bipolar pulses of durations 2 and 3 ns has been elaborated and conditions for the stable operation of gas gaps of independent channels without external control pulses have been determined. Each element of the 2 × 2 array of combined antennas is driven from an individual channel of the pulse former. Antennas excited by pulses of the same duration are arranged diagonally. Two radiation synthesis modes have been examined: one aimed to attain ultimate field strength and the other aimed to attain an ultimate width of the radiation spectrum. The modes were changed by changing the time delay between the 2-ns and 3-ns pulses. For the first mode, radiation pulses with a frequency band of 0.2-0.8 GHz and an effective potential of 500 kV have been obtained. The synthesized radiation pulses produced in the second mode had an extended frequency band (0.1-1 GHz) and an effective potential of 220 kV. The pulse repetition frequency was 100 Hz.

A source of high-power pulses of elliptically polarized ultrawideband radiation.

Here, we describe a source of high-power ultrawideband radiation with elliptical polarization. The source consisting of a monopolar pulse generator, a bipolar pulse former, and a helical antenna placed into a radioparent container may be used in tests for electromagnetic compatibility. In the source, the helical antenna with the number of turns N = 4 is excited with a high-voltage bipolar pulse. Preliminary, we examined helical antennas at a low-voltage source aiming to select an optimal N and to estimate a radiation center position and boundary of a far-field zone. Finally, characteristics of the source in the operating mode at a pulse repetition rate of 100 Hz are presented in the paper as well. Energy efficiency of the antenna is 0.75 at the axial ratio equal to 1.3. The effective potential of radiation of the source at the voltage amplitudes of the bipolar pulse generator equal to -175/+200 kV reaches 280 kV.

[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.

[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.

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.

[Hydrodynamics-based transfer of human apolipoprotein A-I gene into mice: study of factors involving an efficacy and duration of the transferred gene expression in animals' liver]. / Perenos gena apolipoproteina A-I cheloveka mysham metodom gidrodinamicheskikh in''ektsii: faktory, vliiaiushchie na éffektivnost' i prodolzhitel'nost' ékspressii gena v pecheni zhivotnogo.

Human apolipoprotein A-I gene (apoA-I) plasmid expression vectors were transferred into mice by hydrodynamic injections into tail vein. Two types of expression vectors were used. First one -pCMVcapoAI contains cDNA of apo A-I driven by human cytomegalovirus early gene promoter (CMV). Second one--pAlg contains genomic locus of intron-containing apo A-I under control of own extended 5'-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to appearance of human apo A-I mRNA in the liver and human Apo A-I protein in the serum of injected mice. Dynamics of human Apo A-I content in the serum of mice injected by pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, maximal concentration of human Apo A-I protein in the mouse serum was detected one day after injection with following decline to zero level during next two weeks. Under the same conditions injections of pAlg led to maximal level of human Apo A-I concentration in the mouse serum (up to 20 mkg/ml in some animals) on the 5th-7th day of experiment with following graduate decline during several months (human Apo A-I concentration in the serum of oldest analyzed mouse (6 months after injection) was about 25% of its maximal level in the same animal). Levels of human Apo A-I concentration in the mouse serum were compatible after injections of both expression vectors, in spite of much more strong activity of CMV promoter in comparison with APOAI in cultured human hepatoma cells HepG2. We ascribe the revealed difference in dynamics of human Apo A-I expression to delay of apo A-I transcription from pAlg vector, that was confirmed by nested RT-PCR. Significant level and long-term persistence of human Apo A-I in the serum of mice injected by pAlg could be explained by properties of APOAI or (and) exon-intron structure of genomic apo A-I gene. To test the role of APOAI in long-term expression of human Apo A-I in the mice we performed hydrodynamic injections of plasmid vectors containing cDNA of reporter gene encoding luciferase driven by variants of APOAI. No long-term expression of luciferase was found in the livers of injected mice. Therefore, our data suggest the role of exon-intron structure in maintaining of efficient and long-term expression of transferred human apo A-I.