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.

[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.

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.

[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.

[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.

[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.

[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).

[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).

Effects of low-intensity ultrahigh frequency electromagnetic radiation on inflammatory processes.

Low-intensity ultrahigh frequency electromagnetic radiation (42 GHz, 100 microW/cm(2)) reduces the severity of inflammation and inhibits production of active oxygen forms by inflammatory exudate neutrophils only in mice with inflammatory process. These data suggest that some therapeutic effects of electromagnetic radiation can be explained by its antiinflammatory effect which is realized via modulation of functional activity of neutrophils in the focus of inflammation.

[Decrease in the intensity of the cellular immune response and nonspecific inflammation upon exposure to extremely high frequency electromagnetic radiation]. / Snizhenie intensivnosti kletochnogo immunnogo otveta i nespetsificheskogo vospaleniia pri deistvii élektromagnitnogo izlucheniia kraine vysokikh chastot.

The effect of low-intensity extremely high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.1 mW/cm2, 20 min daily) on cell-mediated immunity and nonspecific inflammatory response in mice was studied. The intensity of cell-mediated immune response in the reaction of delayed-type hypersensitivity and nonspecific inflammation was estimated by a relative increase in the thickness of foot pad after immunization of animals by sheep red blood cells or zymosan. It was shown for the first time that the radiation reduces both immune and nonspecific inflammatory responses. It was shown with the use of models of acute inflammation and full-thickness skin wounds that EHF EMR suppresses the nonspecific inflammatory response but does not influence the duration of the pathological process. We suppose that the basis of the effects revealed is the modification of functional activity of phagocytic cells under the influence of EHF EMR. The results suggest that some therapeutic effects of EHF EMR can be realized via the inhibition of inflammatory processes.

[Effects of low-intensity extremely high frequency electromagnetic radiation on chromatin structure of lymphoid cells in vivo and in vitro]. / Vliianie nizkointensivnogo krainevysokochastotnogo élektromagnitnogo izlucheniia na strukturu khromatina limfoidnykh kletok in vivo i in vitro.

Using a comet assay technique, it was shown for the first time that low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) in vivo causes oppositely directed effects on spatial organization of chromatin in cells of lymphoid organs. In 3 hrs after single whole-body exposure of NMRI mice for 20 min at 42.0 GHz and 0.15 mW/cm2, an increase by 16% (p < 0.03 as compared with control) and a decrease by 16% (p < 0.001) in fluorescence intensity of nucleoids stained with ethidium bromide were found in thymocytes and splenocytes, respectively. The fluorescence intensity of stained nucleoids in peripheral blood leukocytes was not changed after the exposure. The exposure of cells of Raji hunan lymphoid line and peripheral blood leukocytes to the EHF EMR in vitro induced a decrease in fluorescence intensity by 23% (p < 0.001) and 18% (p < 0.05), respectively. These effects can be determined by changes in a number of physiological alkali-labile sites in DNA of exposed cells. We suggested that the effects of low-intensity EHF EMR on the immune system cells are realized with the participation of neuroendocrine and central nervous systems.

[Suppression of nonspecific resistance of the body under the effect of extremely high frequency electromagnetic radiation of low intensity]. / Podavlenie nespetsificheskoi rezistentnosti organizma pri deistvii krainevysokochastotnogo élektromagnitnogo izlucheniia nizkoi intensivnosti.

The dynamics of leukocyte number and functional activity of peripheral blood neutrophils under whole-body exposure of healthy mice to low-intensity extremely-high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.15 mW/cm2, 20 min daily) was studied. It was shown that the phagocytic activity of peripheral blood neutrophils was suppressed by about 50% (p < 0.01 as compared with the sham-exposed control) in 2-3 h after the single exposure to EHF EMR. The effect persisted for 1 day after the exposure, and then the phagocytic activity of neutrophils returned to the norm within 3 days. A significant modification of the leukocyte blood profile in mice exposed to EHF EMR for 5 days was observed after the cessation of exposures: the number of leukocytes increased by 44% (p < 0.05 as compared with sham-exposed animals), mostly due to an increase in the lymphocyte content. The supposition was made that EHF EMR effects can be mediated via the metabolic systems of arachidonic acid and the stimulation of adenylate cyclase activity, with subsequent increase in the intracellular cAMP level. The results indicated that the whole-body exposure of healthy mice to low-intensity EHF EMR has a profound effect on the indices of nonspecific immunity.

[Dimethyl suberimidate as a specific inductor of apoptosis in transformed cells]. / Dimetilsuberimidat kak spetsificheskii induktor apoptoza transformirovannykh kletochnykh kul'tur.

A modification of protein-protein interactions can be considered to be a way to regulate cell death. Chemical cross-linking agents have been traditionally used for protein complexing. This study has been undertaken to test a possibility to induce and(or) to modify cell death by a homobifunctional cross-linker dimethyl suberimidate (DMS). It was shown that the protein cross-linking by DMS resulted in a death of transformed cells by apoptosis. DMS-induced apoptosis was accompanied by cell cycle perturbations and down-regulation of p21/Waf1 mRNA expression. The RT-PCR analysis of bcl-2 family genes revealed the engagement of mitochondria in DMS-induced cytotoxicity. Then, the influence of DMS treatment on TNF-dependent and Fas-mediated apoptosis was investigated. Cell pre-incubation with DMS resulted in their increasing sensitivity for the TNF cytotoxic effect, though activities of anti-Fas cytotoxic antibodies were inhibited. The effects observed are probably due to cross-linking of TNF-receptors. Thus, this study first demonstrated that a chemical cross-linker DMS in capable of inducing apoptosis in transformed cells and modifying TNF-dependent and Fas-mediated apoptosis.

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.

[Effect of extremely high frequency electromagnetic radiation of low intensity on parameters of humoral immunity in healthy mice]. / Vliianie krainevysokochastotnogo élektromagnitnogo izlucheniia nizkoi intensivnosti na pokazateli gumoral'nogo immuniteta zdorovykh myshei.

The modification of indices of the humoral immune response to thymus-dependent antigen (sheep erythrocytes) after a whole-body exposure of healthy mice to low-intensity extremely-high-frequency electromagnetic radiation was studied. Male NMRI mice were exposed in the far-field zone of horn antenna at a frequency of 42.0 GHz and energy flux density of 0.15 mW/cm2 under different regimes: once for 20 min, for 20 min daily during 5 and 20 successive days before immunization, and for 20 min daily during 5 successive days after immunization throughout the development of the humoral immune response. The intensity of the humoral immune response was estimated on day 5 after immunization by the number of antibody-forming cells of the spleen and antibody titers. Changes in cellularity of the spleen, thymus and red bone marrow were also assessed. The indices of humoral immunity and cellularity of lymphoid organs changed insignificantly after acute exposure and series of 5 exposures before and after immunization of the animals. However, after repeated exposures for 20 days before immunization, a statistically significant reduction of thymic cellularity by 17.5% (p < 0.05) and a decrease in cellularity of the spleen by 14.5% (p < 0.05) were revealed. The results show that low-intensity extremely-high-frequency electromagnetic radiation with the frequency and energy flux density used does not influence the humoral immune response intensity in healthy mice but influences immunogenesis under multiple repeated exposures.

Functional peculiarities of MAP2 in DBA/2J inbred mice as a component of genetic predisposition to seizures.

We compared the content of high molecular microtubule-associated protein-2 and its phosphorylation by cAMP- and Ca(2+)/calmodulin-dependent protein kinases in the brain of DBA/2J and C57Bl/6 inbred mice. The revealed differences in protein content and phosphorylation can be attributed to the mechanisms mediating audiogenic seizures in DBA/2J mice and to differential sensitivity of these inbred lines to seizure-inducing factors.

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.