α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

Synthetic ACTH-Like Peptide GKVLKKRR,Corresponding to the Fragment 81–88 of Human Pro-Interleukin-1α, Acts as an Antagonist of ACTH Receptor

Synthetic adrenocorticotropic hormone (ACTH)-like octapeptide leukocorticotropin (GKVLKKRR), corresponding to the amino acid sequence 81–88 of pro-interleukin-1α, was labeled with tritium (specific activity of 22 Ci/mmol) and was found to bind to rat adrenal cortex membranes with high affinity and specificity (K _d = 2.2 ± 0.1 nM). Synthetic ¹²⁵I-labeled ACTH fragment 11–24 was also obtained (specific activity of 98 Ci/mmol) and shown to bind to ACTH receptor on rat adrenal cortex with high affinity (K _d = 1.8 ± 0.1 nM). Unlabeled leukocorticotropin was found to actively replace ¹²⁵I-labeled ACTH (11–24) in the receptor-ligand complex (K _i = 2.0 ± 0.1 nM). Leukocorticotropin at concentration range of 1–1000 nМ did not affect the adenylate cyclase activity in adrenocortical membranes. Thus, leukocorticotropin is an antagonist of ACTH receptor. ACTH-like peptide GKVLKKRR is an antagonist of ACTH This work financeed by the Russian Foundation for Basic Research (grant No. 05-04-48060), by the programs Leading Scientific Schools (grant No. 312.2003.4), Molecular and Cellular Biology (chairman V.M. Lipkin), and Naukogrady (grant No. 04-04-97200), and by the International Science and Technology Center (project No. 2615). V.V. Yurovsky is supported by the American Heart Association (grant No. 0555415U).     

Properties and mechanism of the action of the synthetic peptide octarphin

The peptide TPLVTLFK, whose amino acid sequence corresponds to the 12–19 fragment of β-endorphin (the author’s name for the peptide octarphin), and its analogues (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, and TPLVTLFL) have been synthesized. Tritium-labeled octarphin (specific activity of 28 Ci/mol) has been obtained, and its binding to murine peritoneal macrophages has been studied. It was found that [³H]octarphin binds to macrophages with a high affinity (K _d 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin to macrophages was inhibited by the unlabeled β-endorphin and the selective agonist of the nonopioid β-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, α-endorphin, γ-endorphin, and [Met5]enkephalin (K _i > 10 μM). The inhibitory activity of the octarphin analogues was more than 100 times lower than that of octarphin. It was shown that octarphin stimulates the activity of mouse immunocompetent cells in vitro and in vivo; at a concentration of 1–10 nM, it increased the adhesion and spreading of peritoneal macrophages and their ability to digest the bacteria of the Salmonella typhimurium virulent strain 415 in vitro. The intraperitoneal injection of the peptide at a dose of 20 μg/animal on day 7, 3, and 1 prior to the isolation of cells led to an increase in the activity of the peritoneal macrophages and the Tand B lymphocytes of the spleen.     

The LKEKK synthetic peptide as a ligand of rat intestinal epithelial cell membranes

A tritium-labeled synthetic LKEKK pentapeptide corresponding to the sequences 16–20 of human thymosin-α₁ and 131–135 of human interferon-α₂ was obtained with a specific activity of 28 Ci/mmol. [³H]LKEKK was found to bind with high affinity (K _d 3.7 ± 0.3 nM) to the membranes isolated from epithelial cells of rat small intestinal mucosa. The trypsin treatment of the membranes did not affect the binding, thus supporting the nonprotein nature of the peptide receptor. The binding of the labeled peptide was inhibited by unlabeled thymosin-α₁, interferon-α₂, and cholera toxin B subunit (K _i 4.2 ± 0.4, 3.5 ± 0.3, and 4.7 ± 0.3 nM respectively). The pentapeptide did not affect the adenylate cyclase activity within the concentration range of 1–1000 nM.     

Stimulation of murine natural killer cells by weak electromagnetic waves in the centimeter range]

Irradiation with electromagnetic waves (8.15-18 GHz, 1 Hz within, 1 microW/cm2) in vivo increases the cytotoxic activity of natural killer cells of rat spleen. In mice exposed for 24-72 h, the activity of natural killer cells increased by 130-150%, the increased level of activity persisting within 24 h after the cessation of treatment. Microwave irradiation of animals in vivo for 3.5 and 5 h, and a short exposure of splenic cells in vitro did not affect the activity of natural killer cells.     

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 (K _d 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) (K _i 2.3 ± 0.2 nM), whose [³H] labeled derivative binds to rat adrenocortical membranes (K _d 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) (K _i 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.     

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.     

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.     

Effect of the B Subunit of the Cholera Toxin on the Raw 264.7 Murine Macrophage-Like Cell Line

Russian Journal of Bioorganic Chemistry - The 125I-labeled B-subunit of the cholera toxin ([125I]CT-B with specific activity 98 Ci/mmol) was found to be bonded to the murine macrophage-like cells...