V. B. Sadovnikov

Binding of synthetic fragments of β-endorphin to nonopioid β-endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [3H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (Kd = 2.0 ± 0.1 nM). The [3H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (Ki = 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met5]enkephalin (Ki > 10 µM). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [3H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the authors name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (Ki = 3.1 ± 0.3 nM). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [3H]Octarphin was found to bind to macrophages with high affinity (Kd = 2.3 ± 0.2 nM). The specific binding of [3H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (Ki = 2.4 ± 0.2 and 2.7 ± 0.2 nM, respectively). Copyright

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-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 (Kd = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (Ki = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met5]enkephalin (Ki > 10 μM). 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 immuno-competent 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.

Binding of synthetic peptide TPLVTLFK to nonopioid beta-endorphin receptor on rat brain membranes.

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (Kd = 2.6 ± 0.2 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met5]enkephalin, and [Leu5]enkephalin, as well. The [3H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (Ki = 2.4 ± 0.2 nM) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (Ki = 2.9 ± 0.2 nM). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [3H]immunorphin with membranes (Ki = 2.8 ± 0.2 nM). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright

[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.

Synthetic peptide octarphin (TPLVTLFK), a selective agonist of nonopioid β‐endorphin receptor, stimulates nitric oxide synthesis in macrophages

Synthetic peptide octarphin (TPLVTLFK, a selective agonist of nonopioid β‐endorphin receptor) was able to activate in a dose‐dependent manner murine macrophages to express nitric oxide (NO) synthase and to produce NO. Octarphin required lipopolysacharide for the optimal induction of NO production. Octarphin‐dependent NO production was sensitive to inhibition by dexamethasone and the NO synthase specific inhibitor NG‐monomethyl‐l‐arginine. In the concentration range of 1–1000 nM, octarphin increased the cyclic 3′,5′‐guanosine monophosphate (cGMP) content in macrophages stimulated with lipopolysacharide. The effect was dependent on the peptide concentration and was maximal at a concentration of 100 nM. Thus, octarphin stimulates both NO and cGMP production in macrophages. Copyright

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 (Kd 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 (Ki 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.

Comparative Study of Anti-Inflammatory Effects of Low-Intensity Extremely High-Frequency Electromagnetic Radiation and Diclofenac on Footpad Edema in Mice

Anti-inflammatory effects of low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) was compared to the action of sodium diclofenac on zymosan-induced footpad edema in NMRI mice. Diclofenac in doses of 2, 3, 5, 10, and 20 mg/kg was injected intraperitoneally (ip) 30 min after zymosan injection. The mice were whole-body exposed to EHF EMR (42.0 GHz, 0.1 mW/cm2) for 20 min at 1 h after zymosan injection. Inflammation was assessed over 3–8 h after initiation by measuring the footpad edema and hyperthermia of the inflamed paw. Diclofenac caused a dose-dependent anti-inflammatory effect. Doses of 5–20 mg/kg reduced the footpad edema on the average by 26% as compared to the control. Hyperthermia decreased with increasing in a dose of diclofenac, and at a dose of 20 mg/kg decreased by 60% compared to control. EHF EMR reduced both the footpad edema and hyperthermia by about 20% that was comparable with the effect of single therapeutic dose of diclofenac (3–5 mg/kg). Combined action of diclofenac and EHF EMR exposure caused a partial additive effect. The results obtained suggest that the adjunctive use of low-intensity EHF EMR with non-steroid anti-inflammatory drugs can provide more pronounced therapeutic effects.

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.

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 μg/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 abrupt decrease in its activity. The peptide analogues exhibit a low stress-protective activity and had a low affinity for the ACTH receptor.

α 1 -Thymosin, α 2 -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 (Kd = 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 (Ki > 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.