[Antioxidative and detoxifying effects of analogues of delta-sleep inducing peptide (DSIP)].

16 DSIP analogues with substitutions of 1-2 amino acid residues were synthesized in order to investigate their potential use in medicine. Antioxidative properties of these peptides were studied in vitro and their detoxifying activity was examined in vivo on a model of toxicosis that was induced by the cisplatin cytostatic, which has been widely used in the cancer treatment. Practically all the studied DSIP analogues were shown to exhibit considerable direct antioxidative activity (AOA), and that of the ID-6 analogue was higher than AOA of DSIP and comparable with AOA of vitamin C and ß-carotine. This analogue also demonstrated the most pronounced detoxifying effect towards cisplatin action, resulting in a decrease in the animal death from the acute cisplatin toxicity to 17% (in comparison with 50-67% for the control animals) and restoration of a number of cisplatin-sensitive biochemical blood parameters: decrease in the activity of aspartate aminotransferase and alanine aminotransferase and downregulation of the concentration of the final products of nitrogen exchange (creatinine and urea). Thus, the DSIP-relative peptides could be promising agents for the decrease in the toxic effects of cytostatics that are used in oncology.

[IL-2-receptor associated action of the modified peptide fragments of human IL-2 on macrophages].

Synthetic peptides corresponding to the 59-72 (I), 60-72 (II) and 61-72 (III) sequences of human interleukin 2 with their N(alpha) acetylated and C(alpha) methylated termini were shown to exhibit pronounced hepatoprotective properties. These peptides neutralized hepatotoxic effects of such agents as tetrachloromethane and galactosamine in experiments in vivo. The peptide action revealed as normalization of duration of the thiopental narcosis of experimental animals and the level of hepatospecific enzymes in their blood. The effects of peptides (I)-(III) proved to be similar to that of prednisolone (the well-known anti-inflammatory agent), whereas the bestatine cytotoxic dipeptide had no hepatoprotecting effect. The target of the hepatoprotective activity of the peptides was shown to be the preliminary activated macrophages. We proposed that this activity of the peptides was associated with their interaction with the a-subunit of the interleukin 2 receptor (IL-2Ralpha), because the X-Ray analysis pointed to this region as one of binding sites of IL-2 with IL-2Ralpha. Experiments on the influence of the most active (59-72)-peptide on growth of the IL-2 dependent cell line (CTLL) confirmed this proposal. The 3H-labeled peptide corresponding to the 59-72 sequence ofthe human IL-2 was shown to bind to the CTLL cels. We assumed that the binding of this peptide was specific and occurred precisely with IL-2Ra and virtually determined the binding constant. Its value (1.41 x 10(-6) M) was comparable with that of the interaction of IL-2 with IL-2Ralpha (approximately 10(-7) M).

[Molecular mechanisms regulating the activity of macrophages].

This article reviews modern concepts of the most common types of macrophage activation: classical, alternative, and type II. Molecular mechanisms of induction and regulation of these three types of activation are discussed. Any population of macrophages was shown to change its properties depending on its microenvironment and concrete biological situation (the "functional plasticity of macrophages"). Many intermediate states of macrophages were described along with the most pronounced and well-known activation types (classical activation, alternative activation, and type II activation). These intermediate states are characterized by a variety of combinations of their biological properties, including elements of the three afore mentioned types of activation. Macrophage activity is regulated by a complex network of interrelated cascade mechanisms.

Study of secondary specificity of enteropeptidase in comparison with trypsin.

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu)n-Lys(Arg)-(downward arrow)-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM(-1) x min(-1)) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM(-1) x min(-1)).

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide.

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

The synthetic peptide related to the central part of human interleukin-2 molecule accelerates growth and vascularization of sarcoma 180 in mice.

The synthetic peptide C-1-6 related to the central part of human interleukin 2 molecule (sequence 59-72; N- and C-modified) had been shown previously to inhibit cytotoxic activity of macrophages converting them to synthesis of growth factors. In this paper the effect of C-1-6 on growth of sarcoma 180 in mice was studied. C-1-6 significantly accelerated tumor growth having been injected into mice in dose 5 or 50 microg per animal since the 4th day after tumor cells transplantation. Supernatants of Mphi in vitro activated by C-1-6 (10 microg/ml) and injected into mice also accelerated significantly sarcoma mass diurnal increasing as compared to mice treated with supernatants of non-activated Mphi or activated with bacterial lipopolysaccharide. A single injection of C-1-6 into mice either at the day or at the next day of tumor cells inoculation increased significantly the number of vessels growing up to transplant, thus the forming of the vascular bed had preceded tumor volume enlargement.

[Isolation, crystallization, and preliminary x-ray study of a Fab fragment of monoclonal antibody against human interleukin-2 and its complex with antigenic peptide]. / Poluchenie, kristallizatsiia i predvaritel'nye rentgenostrukturnye issledovaniia Fab-fragmenta monoklonal'nogo antitela k interleikinu-2 cheloveka i ego kompleksa s antigennym peptidom.

Antigen-binding fragments (Fab) of mouse monoclonal antibodies to human interleukin-2 were obtained in preparative quantities by a modified procedure. These Fab-fragments were shown to be homogeneous according to the isoelectric focusing method. Various monocrystals of these free Fab-fragments and their complexes with the antigenic peptide corresponding to the 59-72 sequence of interleukin-2 were obtained. These were shown to be suitable for X-ray and were preliminarily studied by X-ray.

[The embryotoxic action of fragment C-I-6 of human interleukin-2]. / Embriotoksicheskoe deistvie fragmenta C-I-6 interleikina-2 cheloveka.

The peptide C-I-6 is a modified fragment (sequence 59-72) of human interleukin-2 and stimulates regeneration of hepatic cells and tissues. Study of the possible embryotoxic and teratogenic properties of C-I-6 showed that in some cases it induces the following statistically significant changes: reduces the number of viable fetus, increases the occurrence of hydronephrosis and the number of external and internal hematomas, increases the weight and size of the embryo. It is concluded that intraperitoneal administration of C-I-6 causes a mild embryotoxic and teratogenic effect in rats.

[Immunomodulating properties of synthetic fragments of human interleukin-2]. / Immunomoduliruiushchie svoistva sinteticheskikh fragmentov interleikina-2 cheloveka.

For the study of the antigenic structure and function of human interleukin 2, the peptides corresponding to its 60-72 sequence were synthesized by conventional methods of peptide chemistry in solution. To enhance the stability of the synthetic peptides towards the proteolysis and to remove their terminal charges, we acetylated their NH2 groups and esterified with methanol their carboxyls. Some of these peptides were converted from being cytotoxic to possessing strong growth-stimulating activity for the preliminary activated macrophages both in vitro and in vivo. The biologically active peptides were also shown to enhance regeneration-reparation processes in liver and skin.

[Conformation of fragment 66-72 of interleukin-2 complexed with a monoclonal antibody to interleukin-2]. / Konformatsiia fragmenta 66-72 interleikina-2 v komplekse s monoklonal'nym antitelom k interleikinu-2.

1H-NMR spectra of the interleukin-2 synthetic fragment Ac-Leu66-Glu-Glu-Val-Leu-Asn-Leu72-OCH3 in the presence or absence of the monoclonal antibody were analysed. The data obtained are consistent with an extended unordered conformation of the free peptide. Measurements of NOESY cross-peak intensities allowed us to determine the spatial structure of the peptide bound to the antibody. The peptide has an amphiphilic surface with hydrophobic and hydrophilic amino acid side chains clustered on the opposite sides of its alpha-helical-like structure. The hydrophobic and hydrophilic clusters are located on the opposite sides of the bound peptide's surface. The hydrophobic side chains contact the antibody surface, while the hydrophilic ones are oriented into the solvent (T. A. Balashova et al. (1991) Bioorgan. Khim. (USSR), v. 17, p. 1470-1486). Hydrolysis of the methyl ester slowly ocurs in the presence of the antibody. This process does not alter the conformation of the peptide bounded with the antibody, though decreases the peptide's affinity to the antibody.

[Study of the interaction of the monoclonal antibody to human interleukin-2 and its Fab-fragment with synthetic peptides --interleukin-2 fragments by (1)H-NMR]. / Issledovanie metodom (1)H-IaMR vzaimodeistviia monoklonal'nogo antitela k interlekina-2 cheloveka i ego Fab-fragmenta s sinteticheskimi peptidami--fragmentami interleikina-2.

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

[The effect of a synthetic fragment of human interleukin-2 on the growth and vascularization of sarcoma 180 in mice]. / Vliianie sinteticheskogo fragmenta interleikina-2 cheloveka na rost i vaskuliarizatsiiu sarkomy 180 u myshei.

The effect of a modified peptide of the middle part of the human interleukin-2 molecule (C-1-6) on the growth of transplantable sarcoma 180 of mice was studied. C-1-6 injected intraperitoneally in the dose of 0.5, 5 and 50 micrograms 4 times every other day was shown to stimulate tumor growth and enhance angiogenesis induced by the tumor transplanted intracutaneously. Stimulation of tumor growth was most apparent when the agent was given late (on day 4) after transplantation. Supernatants of murine peritoneal macrophages activated by 1 microgram/ml solution of C-1-6 proved capable of stimulating tumor growth, too. It is suggested that the stimulating effect of C-1-6 on tissue regeneration be investigated.

[Synthesis and immunochemical properties of peptides corresponding to sequences 59-72 and 25-36 of human interleukin-2]. / Sintez i immunokhimicheskie svoistva peptidov posledovatel'nosti 59-72 i 25-36 interleikina-2 cheloveka.

Synthesis of peptides corresponding to the 59-72 and 25-36 sequences of human IL-2 is reported. The former peptide, which had been shown to be immunogenic in the protein molecule, was prepared to obtain antipeptide antibodies for isolation and purification of the recombinant IL-2. We located the epitope at the C-terminus of this peptide. In accordance with the IL-2 secondary structure and hydrophilicity profile analysis, the 25-36 fragment was chosen as the potential epitope. The peptides were synthesized by conventional methods in solution, conjugated with a protein carrier, and polyclonal rabbit antisera were obtained. Antibodies against both peptides were shown to be specific to human IL-2 in ELISA. Besides, monoclonal antibodies to IL-2 recognized in ELISA the 59-72 peptide, suggesting the epitope located in this region to be main one in the protein molecule.