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Dendrimers are regular hyperbranched macromolecules. The complex structure of the relaxation spectrum is one of the many features of dendrimers that does not fit into the classical concepts of poly...

A series of amphiphilic charged copolymers of amino acids is synthesized, and their binding to cationic and anionic peptides is studied. The resulting interpolyelectrolyte complexes are additionally stabilized by cationic/anionic polysaccharides using surface layering. The positive effect of coatings on the release rate of peptides and decrease in the cytotoxicity and increase in the stability of the resulting peptide delivery systems in biological media is shown.

Several hybrid systems for oral delivery of a therapeutic peptide have been prepared and investigated. Poly(l-glutamic acidco-d-phenylalanine) and silica sub-microparticles, porous CaCO3 microparticles and Lycopodium clavatum spore capsules were employed as the first level carriers included in sodium alginate granules as the second level carriers. Efficiency of the peptide encapsulation and release rate strongly depended on the nature and structure of the carriers.

Due to their well-defined structure, multivalency, biocompatibility, and low toxicity, lysine dendrimers can be used as safe and efficient nanocarriers for drug and gene delivery. One useful strategy for improving the gene delivery properties of dendrimers is modification with arginine amino acid (Arg) residues. Incorporation of Arg residues could be favorable for the enhancement in transfection efficiency of lysine based dendrimers. In this work, we have synthesized a new second-generation poly-L-lysine dendrimer with repeating units containing two linear Arg residues between neighboring lysine branching points (Lys-2Arg dendrimer) and studied its physicochemical properties. We confirmed the structure of Lys-2Arg dendrimer using various one- and two-dimensional 1H and 13C NMR spectroscopy methods. Comparison of T1H relaxation data for Lys-2Arg and Lys-2Lys dendrimers showed that the replacement of double Lys residues with double Arg residues resulted in a sharp decrease in the mobility of methylene groups in side segments and in the main chain of e-Lys inner segments. We suggest that this unexpected effect is caused by a guanidine–guanidine pairing effect in water, which leads to entanglements between dendrimer branches.

This paper is aimed at the development of a biosensor for direct detection of Hepatitis C virus (HCV) surface antigen: envelope protein (E2). A recombinant LEL fragment of biological cell receptor CD81 and two short synthetic peptides imitating the fragment of LEL sequence of CD81 (linear and loop-like peptides) capable of specific binding to E2 were tested as molecular recognition elements of the biosensor. For this purpose the selected ligands were immobilized to the surface of a screen-printed electrode utilized as an electrochemical sensor platform. The immobilization parameters such as the ligand concentration and the immobilization time were carefully optimized for each ligand. Differential pulse voltammetry used to evaluate quantitatively binding of E2 to the ligands revealed their similar binding affinity towards E2. Thus, the linear peptide was selected as a less expensive and easily prepared ligand for the HCV biosensor preparation. The resulting HCV biosensor demonstrated selectivity towards E2 in the presence of interfering protein, conalbumin. Moreover, it was found that the prepared biosensor effectively detected E2 bound to hepatitis C virus-mimetic particles (HC VMPs) at LOD value of 2.1∙10−5 mg/mL both in 0.01 M PBS solution (pH 7.4) and in simulated blood plasma.

The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra-and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy. �� 2020 by the authors.

We propose stratified graphene-dielectric structures as a route to active hyperbolic metamaterials. Employing an exact in RPA, non-local quantum conductivity model allows us to describe the graphene plasmons in the photo-inverted graphene carrier system at large wavevectors, which is of key interest for hyperbolic metamaterials. By calculating Bloch modes of these structures, we predict the possibility of mode loss compensation and amplification, as well as show the tunability of these properties.

Due to their excellent biocompatibility and biodegradability, polypeptides have emerged as versatile bioinspired scaffolds for the preparation of artificial biomaterials. In order to create self-assembled polypeptide nanoparticles with enhanced stability towards enzymatic degradation, we synthesized a series of random and block polypeptides based on lysine and alpha-aminoisobutyric acid (Aib) by the ring-opening polymerization of N-carboxyanhydrides (ROP NCA) of the corresponding amino acids. A conformational analysis carried out by means of FT-IR absorption and CD spectroscopies revealed a noticeable difference between random and block copolymers. In turn, the spatial organization of the polypeptide chains induced the formation of nanostructures of different types. The block copolymers self-assembled in vesicle-like structures, whereas polypeptides with randomly distributed monomers formed micelles. In contrast with the polymers with only natural amino acids, all nanoparticles based on Aib/Lys polypeptides showed strong resistance to proteolytic cleavage. The cytotoxicity and the kinetics of the cellular uptake of the prepared nanostructures were also studied. The results obtained could not only contribute to the understanding of long Aib polypeptide folding and self-assembling, but also pave the way to the design of nanomaterials with finely tuned properties in the fields of drug delivery and tissue engineering.

The development and application of novel nanospheres based on cationic and anionic random amphiphilic polypeptides with prolonged stability were proposed. The random copolymers, e.g., poly(l-lysine-co-d-phenylalanine) (P(Lys-co-dPhe)) and poly(l-glutamic acid-co-d-phenylalanine) (P(Glu-co-dPhe)), with different amount of hydrophilic and hydrophobic monomers were synthesized. The polypeptides obtained were able to self-assemble into nanospheres. Such characteristics as size, PDI and &zeta, potential of the nanospheres were determined, as well as their dependence on pH was also studied. Additionally, the investigation of their biodegradability and cytotoxicity was performed. The prolonged stability of nanospheres was achieved via introduction of d-amino acids into the polypeptide structure. The cytotoxicity of nanospheres obtained was tested using HEK-293 cells. It was proved that no cytotoxicity up to the concentration of 500 &micro, g/mL was observed. C-peptide delivery systems were realized in two ways: (1) peptide immobilization on the surface of P(Glu-co-dPhe) nanospheres, and (2) peptide encapsulation into P(Lys-co-dPhe) systems. The immobilization capacity and the dependence of C-peptide encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The kinetic of drug release was studied at model physiological conditions. Novel formulations of a long-acting C-peptide exhibited their effect ex vivo by increasing activity of erythrocyte Na+/K+-adenosine triphosphatase.

Among the various biocompatible amphiphilic copolymers, biodegradable ones are the most promising for the preparation of drug delivery systems since they are destroyed under physiological conditions, that, as a rule, reduce toxicity and provide controlled release of the drug. Hybrid graft-copolymers consisting of the main inorganic polyphosphazene chain and polypeptide side chains are of considerable interest for the development of delivery systems with a controlled degradation rate, since the main and side chains will have different degradation mechanisms (chemical and enzymatic hydrolysis, respectively). Variable particle degradation rate, controlled by the adjusting the composition of substituents, will allow selective delivery in vivo and controlled drug release. The present work proposes the preparation of biodegradable macroinitiators based on polyorganophosphazenes for the synthesis of hybrid copolymers. Synthesis of novel biodegradable macroinitiators based on polyorganophosphazenes was performed via macromolecular substitution of a polydichlorophosphazene chain with the sodium alcoholates, amines and amino acids. The composition of copolymers obtained was calculated using NMR. These polyorganophosphazenes bearing primary amino groups can be considered as convenient macroinitiators for the polymerization of NCA of α-amino acids in order to prepare hybrid copolymers polyphosphazene-graft-polypeptide. The developed macroinitiators were amphiphilic and self-assembled in the aqueous media into nanoparticles. Furthermore, the ability to encapsulate and release a model substance was demonstrated. In addition, the in vitro cytotoxicity of synthesized polymers was evaluated using two cell lines.

Polymyxins are peptide antibiotics that are highly efficient against many multidrug resistant pathogens. However, the poor stability of polymyxins in the bloodstream requires the administration of high drug doses that, in turn, can lead to polymyxin toxicity. Consequently, different delivery systems have been considered for polymyxins to overcome these obstacles. In this work, we report the development of polymyxin delivery systems based on nanoparticles obtained from the self-assembly of amphiphilic random poly(L-glutamic acid-co-D-phenylalanine). These P(Glu-co-DPhe) nanoparticles were characterized in terms of their size, surface charge, stability, cytotoxicity, and uptake by macrophages. The encapsulation efficiency and drug loading into P(Glu-co-DPhe) nanoparticles were determined for both polymyxin B and E. The release kinetics of polymyxins B and E from nanoformulations was studied and compared in buffer solution and human blood plasma. The release mechanisms were analyzed using a number of mathematical models. The minimal inhibitory concentrations of the nanoformulations were established and compared with those determined for the free antibiotics. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

In order to enhance intracellular uptake and accumulation of therapeutic nucleic acids for improved gene therapy methods, numerous delivery vectors have been elaborated. Based on their origin, gene carriers are generally classified as viral or non-viral vectors. Due to their significantly reduced immunogenicity and highly optimized methods of synthesis, nanoparticles (especially those imitating natural biomolecules) constitute a promising alternative for virus-based delivery devices. Thus, we set out to develop innovative peptide dendrimers for clinical application as transfection agents and gene carriers. In the present work we describe the synthesis of two novel lysine-based dendritic macromolecules (D3K2 and D3G2) and their initial characterization for cytotoxicity/genotoxicity and transfection potential in two human cell line models: cervix adenocarcinoma (HeLa) and microvascular endothelial (HMEC-1). This approach allowed us to identify more cationic D3K2 as potent delivery agent, being able to increase intracellular accumulation of large nucleic acid molecules such as plasmids. Moreover, the dendrimers exhibited specific cytotoxicity towards cancer cell line without showing significant toxic effects on normal cells. These observations are promising prognosis for future clinical application of this type of nanoparticles.

Peptide dendrimers are good candidates for diverse biomedical applications due to their biocompatibility and low toxicity. The local orientational mobility of groups with different radial localization inside dendrimers is important characteristic for drug and gene delivery, synthesis of nanoparticles, and other specific purposes. In this paper we focus on the validation of two theoretical assumptions for dendrimers: (i) independence of NMR relaxations on excluded volume effects and (ii) similarity of mobilities of side and terminal segments of dendrimers. For this purpose we study 1H NMR spin-lattice relaxation time, T1H, of two similar peptide dendrimers of the second generation, with and without side fragments in their inner segments. Temperature dependences of 1/T1H in the temperature range from 283 to 343 K were measured for inner and terminal groups of the dendrimers dissolved in deuterated water. We have shown that the 1/T1H temperature dependences of inner groups for both dendrimers (with and without side fragments) practically coincide despite different densities of atoms inside these dendrimers. This result confirms the first theoretical assumption. The second assumption is confirmed by the 1/T1H temperature dependences of terminal groups which are similar for both dendrimers.

The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

Well-defined homopolymers of 2-deoxy-2-methacrylamido- d -glucose (MAG) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization using 4-cyanopentanoic acid-4-dithiobenzoate as chain transfer agent (CTA) and 2,2′-azobisisobutyronitrile (AIBN) as initiator. The effect of polymerization conditions such as molar ratio of RAFT agent to initiator and monomer to RAFT agent on conversion and molecular mass characteristics of prepared polymers was estimated. Kinetics of the polymerization under various reaction conditions was studied as a function of time and conversion using in situ 1H NMR spectroscopy. The living character of polymerization was demonstrated by carriyng out the further chain extension experiments using isolated PMAG-CTA as a macroRAFT agent. For that, the PMAG with higher molecular weight, as well as block copolymer with acrylic acid were synthesized and characterized using 1H NMR and SEC analysis. A novel amphiphilic copolymer representing a hybrid of glycopolymer and polypeptide fragments was synthesized using RAFT and ring-opening polymerization (ROP). In this case, dithiobenzoate end-group of PMAG was transformed via simultaneous reduction and in situ thiol capping reaction by cysteamine hydrochloride into amino functionality suitable for polymerization of N-carboxyanhydrides (NCA). Two block copolymers of MAG with poly- l -phenylalanine (PPhe) differed with hydrophobic block length were synthesized, characterized and used for preparation of particles via copolymer self-assembly. The hydrodynamic diameter, morphology and cytotoxicity of polymer particles based on PMAG-b-PPhe were evaluated using DLS, TEM and MTT-assay, respectively. Moreover, the encapsulation of model compound (fluorescent dye rhodamine 6G) inside PMAG-b-PPhe micelles, as well as its release, were also tested.

The aim of this work was to study the effect of multiple (during 12 months) immunization of rats with BSA-conjugated peptide 189-205 corresponding to the second extracellular loop of rat HT1BR on ACSS activity in the brain of immunized animals (group HT1BR) and its regulation by hormones.

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

In this research poly(l-lysine)-b-poly(l-leucine) (PLys-b-PLeu) polymersomes were developed. It was shown that the size of nanoparticles depended on pH of self-assembly process and varied from 180 to 650nm. The biodegradation of PLys-b-PLeu nanoparticles was evaluated using in vitro polypeptide hydrolysis in two model enzymatic systems, as well as in human blood plasma. The experiments on the visualization of cellular uptake of rhodamine 6g-loaded and fluorescein-labeled nanoparticles were carried out and the possibility of their penetration into the cells was approved. The cytotoxicity of polymersomes obtained was tested using three cell lines, namely, HEK, NIH-3T3 and A549. It was shown that tested nanoparticles did not demonstrate any cytotoxicity in the concentrations up to 2mg/mL. The encapsulation of specific to colorectal cancer anti-tumor drug irinotecan into developed nanocontainers was performed by means of pH gradient method. The dispersion of drug-loaded polymersomes in PBS was stable at 4°C for a long time (at least 1month) without considerable drug leakage. The kinetics of drug release was thoroughly studied using two model enzymatic systems, human blood serum and PBS solution. The approximation of irinotecan release profiles with different mathematical drug release models was carried out and allowed identification of the release mechanism, as well as the morphological peculiarities of developed particles. The dependence of encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The maximal drug loading was found as 320±55μg/mg of polymersomes. In vitro anti-tumoral activity of irinotecan-loaded polymersomes on a colon cancer cell line (Caco-2) was measured and compared to that for free drug.

The specific activity of the thyroid gland is regulated by thyroid-stimulating hormone (TSH) via TSH receptor (TSHR). This receptor is coupled with various types of G proteins, including Gs proteins, through which TSH stimulates activity of enzyme adenylyl cyclase (AC). Since the use of TSH in medicine is restricted, selective regulators of TSHR with activity of agonists and antagonists are being developed. One approach to their creation is development of peptides corresponding to the functionally important TSHR regions that are located in its cytoplasmic loops and are involved in binding and activation of G proteins. We synthesized peptide corresponding to the C-terminal region 612–627 of the third cytoplasmic loop of TSHR and its derivatives modified by residues of palmitic acid (from the N- or C-termini) or by polylysine dendrimer (from the N-terminus) and we studied their effect on the basal and TSH-stimulated AC activity in membrane fractions isolated from the rat thyroid gland. The most active was peptide 612–627-K(Pal)A modified by palmitate from the C-terminus, where the hydrophobic transmembrane region is located in TSHR. At the micromolar concentrations, it increased AC activity and reduced the AC-stimulating TSH effect. The action of 612–627-K(Pal)A was directed to its homologous TSHR, which is indicated by the following facts: the ingibition of Gs proteins, the transductory component of the AC system, by treatment of the membranes with cholera toxin blocked the AC effect of peptide and this effect was not revealed in the tissues where TSHR are absent, the peptide did not influence the stimulating AC effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer had a much lower ability to activate AC in the thyroid gland than 612–627-K(Pal)A, whereas the peptide modified by palmitate from the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable with 612–627-K(Pal)A by the ability to inhibit the AC action of TSH, but it also decreased (although to the lesser degree) the AC effects of other hormones, which indicates its low receptor specificity. Thereby, the obtained data indicate the high efficiency of the peptide 612–627-K(Pal)A as a regulator of TSHR and the possibilities of creation of drugs on its basis for regulation of thyroid-gland functions in the case of pathology.

Peptide dendrimers, due to their biocompatibility and low toxicity, are highly promising candidates as nanocarriers for drugs and genes. The development of this kind of delivery system requires reliable monitoring of their metabolic and biological pathways. In this respect, hydrogen isotope labeling has tremendous importance, being a safe tool for detection of the labeled nanocarriers. In this work, we have synthesized new histidine-rich lysine-based dendrimers (Lys-2His dendrimer) with two linear histidine (His) residues in every inner segment. The presence of His residues has enabled us to perform controlled deuteration of Lys-2His dendrimers. The high deuteration degree (around 70%) does not practically change after redissolving the samples in H2O and heating them at 40 &deg, C, which indicates the isotopic label stability.

We and other authors have shown that synthetic peptides corresponding to regions of the third cytoplasmic loop (CL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and triggering them in the absence of a hormone. To create selective regulators of hormonal signaling systems on the basis of these peptides, the relationship between their biological activity and secondary structure is studied. It is suggested that the most suitable is the helical conformation, which allows the peptide to effectively interact with signaling proteins. The goal of this study was to test the biological activity and secondary structure of linear peptides that we synthesized and their dimeric and palmitoylated analogs corresponding to the C-terminal region of CL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine (serotonin) receptor of type 6 (Ser6R). It is shown that LHR peptides at micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in plasma membranes of rat testes, while Ser6R peptides activate AC and G-proteins in synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while Ser6R peptides, the effect of Ser6R-agonist, EMD-386088, and the action of the peptides was not found in the case of nonhomologous receptors. Using circular dichroism spectroscopy, it is shown that in the neutral (pH 7) and acidic (pH 2) medium, all the peptides exist predominantly in the antiparallel β-sheet (37–42%) and disordered conformations (33–35%). In the alkaline medium (pH 10) in the case of palmitoylated peptides the increase of the contribution of the helical conformation to 12–27% was observed. In the presence of trifluoroethanol (10–80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs by 14%); however, in this case, the antiparallel β-sheet and disordered conformation prevailed. The conclusion was drawn that the lack of a clearly expressed ability to form helices in peptides derived from CL-3 of receptors did not significantly affect their activity. This is consistent with the proposed mechanism of peptide action, whereby peptide interacts with the complementary regions of homologous receptor that does not require helix formation.

Proximal regions of the third intracellular loop (ICL-3) are responsible for the interaction with heterotrimeric G proteins in most of the serpentine type receptors. The peptides corresponding to these regions are able to activate G proteins in the absence of hormone and to alter the transduction of hormonal signal via the respective homologous receptor. However, the molecular mechanisms of action of the peptides, their specificity to receptors and target tissues are currently not well understood. The goal of this work was to study the receptor and tissue specificity of peptides-derivatives of C-terminal regions of the ICL-3 of luteinizing hormone receptor (LHR), type 1 relaxin receptor (RXFP1), somatostatin receptors of types 1 and 2 (Som1R and Som2R), and 5-hydroxytryptamine receptors of subtype 1B and type 6 (5-HT1BR and 5-HT6R) on the functional activity of adenylyl cyclase (AC) and GppNHp-binding of G proteins in the brain, myocardium, and testis of rats. It was shown that the influence of peptides on AC and G proteins is well detected in tissues enriched in homologous receptors. The effects stimulating AC and GppNHp-binding were most pronounced in the testes for LHR peptide, in the brain for peptide 5-HT6R, and in all of the tested tissues (but mainly in the myocardium) for the RXFP1 peptide. The AC-inhibiting effects of peptides Som1R, Som2R and 5-HT1BR, as well as the stimulation of GppNHp binding induced by these peptides, were most pronounced in the brain. In the presence of the peptides, the AC effects of hormones acting via homologous receptors were significantly attenuated, while the AC effects of other hormones changed insignificantly. The findings suggest that biological activity of the peptides depends on their interaction with complementary regions of homologous receptors, which should be taken into account when developing highly selective regulators of hormonal signaling systems on the basis of these peptides.

A leading role in the interactions of most serotonin-type hormone receptors with heterotrimeric G proteins is played by their third cytoplasmic loops. Studies in recent years have shown that synthetic peptides corresponding to the membrane-proximal parts of these loops may have selective influences on the transmission of hormone signals via their homologous receptors and trigger the signal cascade in the absence of hormone. We report the first synthesis of peptides derived from the C-terminal region of the third cytoplasmic loop of type 1B serotonin receptors, along with studies of their influences on the serotonin sensitivity of the adenylate cyclase system in the rat brain. Peptides 300–316 and 306–316 (numbered from amino acid positions in the rat serotonin subtype 1B receptor molecule) at micromolar concentrations, in the absence of hormone, stimulated GTP-binding Gi-proteins coupled with subtype 1B serotonin receptors and inhibited forskolin-stimulated adenylate cyclase activity. Studies using selective serotonin receptor agonists and antagonists showed that peptides 300–316 and 306–316 inhibit the transmission of the serotonin signal via the homologous receptor but have weak effects on other types of serotonin receptor. Peptide 300–316 were markedly more active than its shortened analog 306–316 in terms of the selectivity and efficacy of actions on the adenylate cyclase signaling system, which is regulated via subtype 1B serotonin receptors. These data provide evidence that region 300–316 of subtype 1B serotonin receptors is involved in the interaction with Gi proteins and includes the main molecular determinants responsible for transmission of the serotonin signal to adenylate cyclase.

Fourier transform infrared spectroscopy (FTIR) was used for determination of orientational and conformational characteristics of plane polypeptide brushes with different density of polypeptide chains grafted to modified silicon surface. The determination of grafting density of polypeptide chains in brushes, which depends on chemical structure of spacer groups, was carried out using ultraviolet (UV) spectroscopy. The influence of chemical structure of modifying spacer groups and outer conditions on the characteristics of plane polypeptide brushes was established. The peculiarities of α-helical structure (random coil transfer of polymer chains in brushes under the action of denaturants) were investigated.

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT1BR) and 6 (5-HT6R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT1BR-peptide ARERKATKTL307–316K-amide mimicking agonist-activated 5-HT1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT1BR-antagonists. The 5-HT6R-peptides mimicking 5-HT6R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT6R-agonists on AC and Gs proteins, but in the presence of 5-HT6R-antagonists their action was blocked. Of all the 5-HT6R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT6R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT1BR and 5-HT6R selectively activate Gi and Gs proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.

The synthesis of polyglutamine conjugates of lysine dendrimers, in which carboxyl groups of polyglutamine chains are linked via one point to the amino groups of N-terminal lysine fragments of dendrimers, is studied. Lysine dendrimers of third, fourth, and fifth generations are preliminarily prepared via the solid-phase synthesis on a polymer support, crosslinked p-methylbenzohydrylaminopolystyrene. The first approach to the synthesis of polyglutamine conjugates of dendrimers consisted in that, after the synthesis of lysine dendrimers, their removal from the polymer support, their full unblocking, and their purification, γ-benzyl glutamate N-carboxyadhydride is involved in solution polymerization at the amino groups of lysine of the outer sphere of dendrimers. The second approach includes the polymerization of γ-benzyl glutamate N-carboxyadhydride on the amino groups of N-terminal lysine residues of lysine dendrimers performed before dendrimer removal from the polymer support. The structural study of star-shaped conjugate of lysine dendrimers makes it possible to for the first time estimate the similarities and differences in these two approaches to the synthesis of polyglutamine conjugates of lysine dendrimers.

To gain insight into the mechanism treating formation of hyperbranched polylysines through the polymerization of N ɛ-carbobenzoxylysine N-carboxyanhydride under conditions of the reductive removal of a N ɛ-carbobenzoxy group, hyperbranched polylysine has been synthesized with the use of trifluoroacetic acid as a terminator in the polymerization of N-carboxyanhydride. The structure of the polymers is studied by capillary electrophoresis, low-pressure gel-permeation chromatography, circular dichroism, and enzymatic hydrolysis with trypsin. At the first stage of synthesis, a low-molecular-mass strongly branched core of the polymer is formed. At the second stage, polylysine chains are grafted via one point onto amino groups of N-terminal lysine moieties of the low-molecular-mass core through their carboxyl ends.

Hydrodynamic and conformational properties of hyperbranched poly(amino acids) based on lysine have been studied by static and dynamic light scattering, velocity sedimentation, translational diffusion, and viscometry in dilute aqueous-saline solutions (0.2 M NaCl). The effects of synthesis conditions of hyperbranched poly(amino acids), modification of their end groups by histidine fragments, and incorporation of diacylated lysine residues between branching points of oligomers and polymers composed of lysine and glutamic acid on the molecular mass characteristics of the hyperbranched polymers have been ascertained. The hydrodynamic properties of the hyperbranched poly(amino acids) differ appreciably from the behavior of both linear polylysine and lysine dendrimers due to conformational features of their macromolecules.

A method for preparing hyperbranched poly(L-lysine) via the polymerization of Nɛ-carbobenzoxy-L-lysine-N-carboxyanhydride has been developed in order to regulate the molecular mass and size of this polymer and to modify amino groups of its N-terminal lysine residues. This method includes the reductive removal of an Nɛ-carbobenzoxy group by hydrogen over activated palladium in the presence of a chain termination agent, which is the activated ether of Nα-tert-butyloxycarbonylhistidine. The structure of the polymers has been studied by capillary electrophoresis, circular dichroism, and molecular hydrodynamics.

The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable to the 612-627-K(Pal)A in the ability to inhibit the AC effect of TSH, but, although to a lesser extent that it decreased the AC effects of other hormones, demonstrating the low receptor specificity. Thus, these data point to the high efficiency of peptide 612-627-K(Pal)A, as a regulator of TSHR, and the prospects of creating the drugs based on it to control the thyroid functions in pathology.

Melanocortin receptors of the type 4 (M4R) play a key role in the regulation of feeding behavior, neuroendocrine functions, and energy metabolism. The alterations in their functional activity induce obesity, metabolic syndrome, depression, and mental disorders, which makes the search of selective regulators of M4R to be one of the actual problems of molecular endocrinology. Promising for the development of such regulators is to design peptides corresponding to functionally important regions of M4R. The purpose of this study was to study the influence of synthesized N-palmitoylated peptide Palm-Thr-Gly-Thr-Ile-Arg-Gln-Gly-Ala-Asn-(Nle)-Lys-Gly-Ala-Ile232-245-amide (Palm-232-245) structurally corresponding to the C-terminal half of the third intracellular loop (ICL-3) of rat M4R on functional activity of adenylyl cyclase signaling system (ACSS) in the fractions of synaptosomal membranes isolated from the brains of male rats. It has been shown that, at a concentration of 10(-7) M and higher, Palm-232-245 stimulates the basal activity of adenylyl cyclase (AC) in the synaptosomal membranes and increases the basal level of GTP binding with the EC50 values of 71 and 267 nM, respectively. Under the combined action of low concentrations of the peptide (10(-7)-10(-6) M) and M4R agonists, α-melanocyte-stimulating hormone (α-MSH) and THIQ (10(-7) M), we observed an additivi stimulatory effect on AC, which disappeared when the peptide concentration was increased to 10(-4)-10(-3) M. In the synaptosomal membranes preincubated with 10(-5) M peptide, the maximum stimulatory effect of M4R agonists on AC activity was lower than that in controls, and EC50 values for this effect, on the contrary, increased. In the case of combined action of the peptide and hormones (γ-MSH, serotonin, PACAP-38) that activate AC via the other receptors, the additivity of their stimulating effects on the ACSS persisted throughout the range of peptide concentrations. The effect of the peptide was not observed in myocardial and testicular membranes no in which there is M4R homologous to the peptide. Thus, N-palmitoylated peptide Palm-232-245 specifically activates the ACSS in the rat brain by acting as intracellular M4R agonist. This may be used to create drugs regulating brain melanocortin system and physiological processes that depend on it.

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(α,e-Lys)4-(α,e-Lys)2-(α,e-Lys)-Ala-NH2 (D1) and ((Lys)8-(α,e-Lys)4-(α,e-Lys)2-α,e-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm) 8 -D1 and (pLys) n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.

We and other authors have shown that synthetic peptides corresponding to regions of the third intracellular loop (ICL-3) of receptors of the serpentine type are capable of activating G-protein signaling cascades and trigger them in the absence of hormone. To create on the basis of these peptides the selective regulators of hormonal signaling systems the relationship between their biological activity and secondary structure are studied. It is assumed that most suitable is a helical conformation, which allows the peptide effectively interact with signaling proteins. The aim of this study was to test the biological activity and secondary structure of synthesized by us linear peptides and their dimeric and palmitoylated analogs, corresponding to C-terminal region of the ICL-3 of luteinizing hormone receptor (LHR) and 5-hydroxytryptamine receptor of the type 6 (5-HT6R). It is shown that LHR-peptides at the micromolar concentrations stimulate the basal activity of adenylyl cyclase (AC) and the GTP-binding of G-proteins in the plasma membranes of rat testes, while 5-HT6R-peptides activate AC and G-proteins in the synaptosomal membranes of rat brain. The action of peptides is tissue-specific and observed in the tissues where there are homologous receptors. The most effective were palmitoylated peptides. LHR-peptide reduced the AC stimulatory effect of human chorionic gonadotropin, while 5-HT6R-peptides the effect of 5-HT6R-agonist, EMD-386088, and the action of the peptides was not found in the case of non-homologous receptors. Using circular dichroism spectroscopy it is shown that in neutral (pH 7) and acidic (pH 2) medium all the peptides are exist predominantly in the antiparallel beta-sheet (37-42%) and disordered conformations (33-35%). In alkaline medium (pH 10) in the case palmitoylated peptides the increase of the contribution of the helical conformation to 12-27% was observed. In the presence of trifluoroethanol (10-80%), a helix-forming solvent, the contribution of helical conformation for the majority of peptides was slightly increased (for palmitoylated analogs to 14%), however, in this case the antiparallel beta-sheet and disordered conformation prevailed. The conclusion was made that the lack of clearly expressed ability to form helices in peptides derived the ICLs of receptors did not significantly affect their activity. This is consistent with proposed mechanism of peptides action, whereby peptide interacts with the complementary regions of homologous receptor that does not require the helix formation.

The third intracellular loops of hormonal receptors play the main role in the interaction of majority of the serpentine type receptors with heterotrimeric G-proteins. In recent years, it was shown that synthetic peptides corresponding to membrane-proximal regions of these loops could be selectively influenced with hormonal signal transduction via the receptors homologous to them and trigger signalling cascade in absence of the hormone. For the first time, we succeeded in synthesizing the peptides derived from C-terminal region of the third intracellular loop of the IB-subtype serotonin receptor and studied their influence on serotonin-sensitive adenylyl cyclase system in the rat brain. The peptides 300-316 and 306-316 (the numbers correspond to amino acid positions in the rat IB-subtype serotonin receptor) at micromolar concentrations in absence of hormone-stimulated GTP-binding of Gi,-proteins coupled with the IB-subtype serotonin receptors and inhibited forskolin-stimulated adenylyl cyclase activity. Using selective agonists and antagonists of serotonin receptors it was shown that the peptides 300-316 and 306--316 inhibited serotonin signal transduction via homologous to them receptor and weakly influenced other types of serotonin receptors. The peptide 300-316 is more active compared with its shorter analogue 306-316 in the selectivity and efficiency of action on adenylyl cyclase signalling system regulated via the IB-subtype serotonin receptors. These findings indicate that the regions 300-316 of the IB-subtype serotonin receptor are involved in interaction with Grproteins and consist of the main molecular determinants responsible for serotonin signal transduction to adenylyl cyclase.

The DNA compacting and transfection properties of hyperbranched polylysines whose N-terminal amino groups were modified with histidine and arginine were studied. The histidine-modified hyperbranched polylysines were shown to provide higher efficacy of binding and transfection in comparison with unmodified or hyperbranched arginine-containing polylysines. This fact was explained by the intrinsic endosomolytic activity of the histidine-modified polymers. The dependence between the quantity of the amino acids that modified the terminal lysine residues in the hyperbranched polylysines, the efficacy of their DNA binding, and the transfection activity of the DNA complexes with the corresponding carriers was found. The possibility to increase the transfection activity of the DNA complexes with the hyperbranched polylysines by glycerin or the JTS-1 amphipathic nonapeptide was studied. At the same time, their simultaneous use was found to result in a transfection decrease.

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for delivering DNA to eukaryotic cells and provide a basis for constructing more refined nonvirus module carriers.

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-NH2 (D1) and (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm)8-D1 and (pLys)n-D1, containing poly(N-vinylimidazole) and polylysine chains bound at a single point to the dendrimer amino groups. The conditions of dendrimer-plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on C2C12 mouse myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

Examinations of the periodontium, carried out in 350 schoolchildren, aged 12 to 16, in the town of Odessa, included registration of the CPITN, PMA, Green-Vermillion's, and CDL indexes. The findings evidence that adolescents aged 12 to 15 have but few periodontal segments with bleeding gingiva, their PMA indexes are reduced, and hygienic status of the oral cavity is getting better, but by the age of 16 accumulation of dental deposit results in inflammation enhancement. Though girls, whatever their age, take better care of the oral cavity, its status is not much better in them than in boys.

This paper discusses the possibilities of using IR Fourier spectroscopy to investigate the orientational and conformational characteristics of single layers of polymers attached by one end to an impermeable surface (polymeric brushes). The method is used to determine the structural parameters of the polymeric branches of brushes attached to a cylindrical or flat surface. © 2005 Optical Society of America

A total of 148 patients aged 18-66 years were examined. Of these, 86 subjects received an experimental diet, while 62 were placed on the common dietetics. Both single and 10-day nutrition with products manufactured in ultra-high ovens (UHF products) did not produce any adverse effect on the mean estimates of acid-forming function of the stomach and pH in the basal phase of gastric secretion or on the pattern of gastric mucus formation in patients. Histological study of the biopsy specimens of the gastric mucosa in patients who received UHF products did not reveal any specific time course of changes in its morphology and function. The blood flow in the gastric mucosa varied within an insignificant range (10-15 cal/min). Fifty per cent of patients who received UHF products without any additional treatment methods showed moderate positive shifts in the clinical and physiological parameters under study. Based on the data obtained the authors draw the conclusion about the lack of an unfavourable effect of UHF products, thinking it advisable to use such products, particularly in a complex of therapeutic measures.