Interaction of Cholera Toxin B-subunit with Human T-lymphocytes.

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1 µM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.

Immunostimulating effect of the synthetic peptide octarphin corresponding to ß-endorphin fragment 12-19.

We have synthesized the peptide TPLVTLFK corresponding to ß-endorphin fragment 12-19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [3H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled b-endorphin and the selective agonist of nonopioid b-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled naloxone, a-endorphin, γ-endorphin, or [Met(5)]enkephalin (K(i) > 10 mM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immunocompetent cells in vitro and in vivo: at concentration of 1-10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 µg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.

[Cardiotropic activity of synthetic peptide CH3CO-Lys-Lys-Arg-Arg-NH2 (protectin)].

Peptide CH3CO-Lys-Lys-Arg-Arg-NH2 (protectin) was synthesized and its activity was studied on the model of experimental myocardial infarction in rats in comparison to the reference antihypoxant drug riboxin. Intranasal injections ofprotectin at doses within 2-20 microg/kg once a day by course of 7 days produced a pronounced anti-ischemic action, improved coronary circulation of the blood, increases contractile activity of myocardium, reduced intensity of lipid peroxidation, and improved antioxidant protection. In some respects (improved coronary circulation of the blood, increased antioxidant protection), protectin was more effective than riboxin.

[Effects and mechanism of action of synthetic peptide octarphin].

We have synthesized the peptide TPLVTLFK corresponding to the ß-endorphin fragment 12-19 (the name given by the authors - octarphin), and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol) and its binding to the murine peritoneal macrophages has been studied. [(3)H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [(3)H]octarphin is inhibited by unlabeled ß-endorphin and selective agonist of non-opioid ß-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM respectively) and not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met(5)]enkephalin (K(i) > 10 µM). Inhibiting activity of unlabeled analogs of octarphin is more then 100 times lower the unlabeled octarphin. Octarphin stimulates activity of murine immunocompetent cells in vitro and in vivo: at the concentration of 1-10 nM enhances the adhesion and spreading of peritoneal macrophages as well as their capacity to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro. Intraperitoneal administration of peptide at dose 20 µg/animal on day 7,3 and 1 prior to the isolation of cells increases activity of peritoneal macrophages as well as T- and B-spleen lymphocytes.

[Changes in the antigenic properties of proteins of laboratory mice during oxidative stress].

Changes in the level of oxidative damage to proteins in CD1 outbred mice gamma irradiated with a dose of 3 Gy have been studied. The changes were estimated from the amount of carbonyl groups (CG) in the proteins. It was found that two hours after exposure to gamma radiation, the amount of CG in the cytoplasmic and nuclear fractions of the liver, heart, brain, and spleen sharply increased. Two months after irradiation, the level of CG in the cytoplasmic and nuclear subcellular fractions of the liver and brain decreased to the level of CG in the control animals, which were not exposed to radiation. In the subcellular fractions of the heart and spleen, the increase in the degree of damage was more significant and a high level of damage was observed even two months after irradiation. An enhancement of the antigenic properties of proteins from the liver, heart, and spleen in the postirradiation period was found. Spleen proteins were most immunogenic. A comparison of the antigenic properties of proteins isolated from the tissues 60 days after irradiation revealed a correlation between the level of oxidative damage and the immunogenicity of the total protein fraction.

[Stress-protective effect of the KKRR synthetic peptide corresponding to the 15-18 sequence of human adrenocorticotropic hormone].

The activity of the KKRR synthetic peptide corresponding to the 15-18 sequence of human adrenocorticotropic hormone (ACTH) and its analogues KKKK, RRRR, RRKK, kKRR, KkRR, KKrR, and KKRr (amino acid residues of the D configuration are designated by small letters) was studied in vivo on rats under cold and heat shock. Intranasal administration of the KKRR peptide at doses of 2-10 microg/animal 1 day before the shock was found to prevent a dramatic increase in the level of corticosterone in rat adrenal glands and blood plasma caused by the temperature effect. Amino acid substitutions in the KKRR peptide were shown to result in an abrupt decrease in its activity. The peptide analogues exhibit a low stress-protective activity and had a low affinity for the ACTH receptor.

[Stress-protective activity of the CH3CO-Lys-Lys-Arg-Arg-NH2 synthetic peptide (protektin)].

The CH3CO-Lys-Lys-Arg-Arg-NH2 peptide (the author has named it protectin) was synthesized, and its activity was studied during different stress actions. Protectin was found to normalize the content of corticosterone and adrenalin in adrenal glands and blood after its intranasal administration to rats one day before a cold or heat shock, or hypobaric hypoxia at doses of 1-10 microg/animal and after its intravenous administration just after acute hemorrhage at doses of 0.5-2 microg/animal. The intranasal administration of protectin at doses of 1-10 microg/rat one day before the heat or cold shock was also shown to prevent a change in the content of free histamine and the activity of diamine oxidase in myocardium, which was induced by the dramatic change in the activity of the enzyme after the temperature actions.

[Antitumor effect of low-intensity extremely high-frequency electromagnetic radiation on a model of solid Ehrlich carcinoma].

The influence of different exposure regimes of low-intensity extremely high-frequency electromagnetic radiation on the growth rate of solid Ehrlich carcinoma in mice has been studied. It was shown that, at an optimum repetition factor of exposure (20 min daily for five consecutive days after the tumor inoculation), there is a clearly pronounced frequency dependence of the antitumor effect. The analysis of experimental data indicates that the mechanisms of antitumor effects of the radiation may be related to the modification of the immune status of the organism. The results obtained show that extremely high-frequency electromagnetic radiation at a proper selection of exposure regimes can result in distinct and stable antitumor effects.

[Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor].

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11-24) and [3H]ACTH (15-18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH (11-24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11-24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15-18) (KKRR) (Ki 2.3 +/- 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 +/- 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15-18) was inhibited by 100% by unlabeled ACTH (11-24) (Ki 2.0 +/- 0.1 nM). ACTH (15-18) in the concentration range of 1-1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.

[Binding of beta-endorphin and its fragments to the non-opiod receptor of murine peritoneal macrophages].

The tritium-labeled selective agonist of the nonopioid beta-endorphin receptor the decapeptide immunorphin ([3H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [3H]immunorphin binds with a high affinity to the non-opioid beta-endorphin receptor of mouse peritoneal macrophages (Kd 2.4 +/- 0.1 nM). The specific binding of [3H]immunorphin to macrophages was inhibited by unlabeled beta-endorphin (Ki of the [3H]immunorphin-receptor complex 2.9 +/- 0.2 nM) and was not inhibited by unlabeled naloxone, alpha-endorphin, gamma-endorphin, and [Met5]enkephalin (Ki > 10 microM). Thirty fragments of beta-endorphin were synthesized, and their ability to inhibit the specific binding of [3H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as beta-endorphin is its fragment 12-19 (Ki 3.1 +/- 0.3 nM).

Role of proteases in activation of apoptosis.

Programmed cell death, or apoptosis, is a physiological cell suicide mechanism, which is triggered in the cells by different stimuli. It has been shown that proteases play a significant role both in the target cell killing by cytotoxic lymphocytes and in the TNF- or anti-Fas-induced cell death. The proteases involved in the early (induction) and late (cell self-destruction) stages of apoptosis are reviewed. It is suggested that the late stages are connected with the activation of a cascade of intracellular proteases, which leads to massive protein destruction. It is likely that the protein destruction is mainly designed for preventing autoimmune response to proteins released from dying cells.

Cell death induced by chemical homobifunctional cross-linkers. Cross-linker induced apoptosis.

Physical association of proteins that underlies cytotoxic signal induction and transduction suggests a possibility of regulating cell response by modifying protein-protein interactions. For protein complexing, chemical cross-linking agents have been traditionally used. However, the ability of various cross-linkers to induce and modify cell responses, cell death in particular, is still obscure. We have undertaken the investigation to test the apoptosis-inducing and modifying properties of the homobifunctional cross-linkers-dimethyl suberimidate (DMS) and 1,5-bis(succinimido-oxycarbonyloxy)pentane (BSOCOP). The functional groups of these cross-linkers are different but both are able to interact with available amino groups. It was shown that bifunctional cross-linkers, unlike their monofunctional analogues, are capable of inducing cell death in transformed cells, thus indicating the crucial role of cross-linking in cell killing. DMS- and BSOCOP-treated cells were shown to undergo cell death by apoptosis, though the signaling pathways were distinct. DMS inhibited bcl-X(L) and bak but not bax gene expression, while BSOCOP potentiated bax mRNA synthesis immediately after application. Cell pre-incubation with DMS, but not with BSOCOP, resulted in an increasing sensitivity to TNF, although activities of anti-Fas cytotoxic antibodies were then inhibited. Thus, this study has demonstrated for the first time that chemical cross-linkers are capable of inducing apoptosis by themselves and modifying the TNF-dependent and Fas-mediated cell death that may have potential therapeutic significance.

Study of protein carbonyls in subcellular fractions isolated from liver and spleen of old and gamma-irradiated rats.

Age- and gamma-irradiation-dependent accumulation of oxidatively modified proteins (measured as carbonyl level) was studied in cytoplasm, mitochondria and nuclei isolated from spleen and liver of 4- and 26-month-old rats. The protein carbonyl levels significantly increased with age in all fractions studied. The carbonyl content was found to be two times higher in the nuclei than in the mitochondria and cytoplasm, which may be related to an extensive modification of lysine and arginine residues in histone molecules. Gamma-Irradiation of rats with 10 Gy caused a rise of protein carbonyls only in their cytoplasm and mitochondria, which was prevented in the animals fed with antioxidants and vitamins for a month before the irradiation. We observed an activation of histone-specific proteases in the nuclei of gamma-irradiated rats. The lack of carbonyl accumulation in the nuclear proteins isolated from tissues of gamma-irradiated animals may be explained by the degradation of oxidized histones by these proteases.

Extracellular diaphorase-like activity as a marker of cytolytic damage to cells.

A new variant of the enzyme release colorimetric method for NK/LAK cytotoxicity is described. Kinetic determination of diaphorase released from damaged target cells (TC) provided an exact measurement of the lytic activity of effector cells (EC). A polyenzyme testing system permitted the use of a wide spectrum of TC. The percentage cytotoxicity and the kinetics of the process coincide, within experimental error, with data from staining with Trypan blue. The method is simple and convenient and can be applied to study cell-mediated cytotoxicity (CMC).

Morphological changes in skin nerves caused by electromagnetic radiation of the millimeter range.

The morphological changes in skin nerves of BALB/C mice after millimeter wavelength range electromagnetic exposure at a frequency of 42.25 GHz and power of 50 mW/cm2 were studied. Immediately after 15 minutes of exposure, the destruction of the cytoplasm of myelinated and unmyelinated axons was found.

Age- and radiation-dependent changes in carbonyl content, susceptibility to proteolysis, and antigenicity of soluble rat liver proteins.

Soluble liver proteins (SLP) from old and gamma-irradiated young rats were studied with respect to their carbonyl content, the rates of autolysis and degradation by proteinase K, and their antigenicity for mice and compared with SLP from non-irradiated young animals. A significant increase in the carbonyl level was found in SLP from old and gamma-irradiated young rats as compared to SLP from intact young rats. The rates of SLP autolysis and proteolysis by proteinase K were increased in the same animal groups but did not correlate the carbonyl level. At the same time, whereas the antigenicity for mice of SLP from old rats was significantly higher than that of SLP from young rats, the antigenicity of SLP from gamma-irradiated rats did not differ from non-irradiated animals. Enrichment of the diet with antioxidant and vitamin supplements (AVS) during one month before the irradiation caused a decrease in the radiation-induced carbonyl level in rat SLP. However, this raised antioxidant level in animal diet did not influence the rates of SLP autolysis and degradation by proteinase K and also did not alter the antigenicity of these proteins. The data allow us to suggest that the increase in autolysis, degradation by the exogenous proteinase, and antigenicity of SLP from old rats are determined not only by carbonyl formation in these proteins due to action of oxygen radicals but also by other age-specific protein modifications.

[Immunochemical study of antigenic determinants of surface protein s of the tropical malaria pathogen Plasmodium falciparum using synthetic peptides]. / Immunokhimicheskoe izuchenie antigennykh determinant poverkhnostnykh belkov vozbuditelia tropicheskoi maliarii Plasmodium falciparum s pomoshch'iu sinteticheskikh peptidov.

To develop new approaches to diagnostics and therapy of malaria, we carried out immunochemical study of the surface proteins of the tropical malaria parasite Plasmodium falciparum with the use of synthetic peptides corresponding to the suggested antigenic determinants of the parasite proteins. Rabbit antisera raised against the synthetic peptides bound to parasite proteins as shown by ELISA and immunoblotting. Affinity purified anti-peptide antibodies inhibited, in some cases, the parasite growth in the human erythrocytes culture.

[The nucleoprotein fraction of mouse serum in normal and pathological processes]. / Issledovanie nukleoproteinovoi fraktsii syvorotki krovi myshei v norme i pri patologicheskikh protsessakh.

Analysis of the blood serum of healthy and infected with malaria plasmodium mice showed a steep rise in content of linear double-stranded DNA (0.2-0.5 and 2-15 gamma/ml, respectively). Some physico-chemical properties of serum DNA and a DNA-associated glycoprotein (M approximately 40 kDa) are determined.

[An ACTH-like peptide immunocortin: effect on the activity of cells from the rat adrenal cortex]. / Vliianie AKTG-podobnogo peptida immunokortina na aktivnost' kletok kory nadpochechnikov krysy.

The effect of immunocortin, an ACTH-like decapeptide VKKPGSSVKV corresponding to the 11-20 sequence of the variable part of the human IgG1 heavy chain on the content of 11-hydroxycorticosteroids (CS) in rat adrenal glands and blood serum in vivo was studied. An intramuscular injection of immunocortin at a dose of 10 microg/kg was found in an hour to induce a twofold decrease in CS content in the adrenal glands and a 1.8-fold increase in the blood serum CS content. At the same time, an immunocortin dose of 100 microg/kg exerted practically no effect on the CS content and its dose of 1000 microg/kg increased the CS content both in adrenal glands and in blood serum by 1.6 and 2.2 times, respectively. Four hours after the injection of any of the three doses of immunocortin, the CS content in adrenal glands did not differ from the control value, and after 24 h the content decreased threefold. Immunocortin was shown to be bound by the ACTH receptors in the membranes of the rat adrenal cortex with a high affinity and specificity (inhibiting the specific binding of 125I-labeled ACTH-(11-24) peptide with Ki of 1.2 nM).

[Production of tumor necrosis factor (TNF) by macrophages and T-lymphocytes of rodents as affected by acute gamma-irradiation]. / Produktsiia faktora nekroza opukholei (FNO) makrofagami i T-limfotsitami gryzunov v usloviiakh ostrogo gamma-oblucheniia.

TNF production in peritoneal macrophages and in splenic T cell from mice and ground-squirrels were performed after acute in vivo and in vitro doses of ionizing radiation ranging from 0.1-6 Gy. It was observed that doses in the range 0.5-6 Gy induced the increase of TNF production along with the decrease of mitogen-induced proliferation in T lymphocytes.