Synthetic Peptide TPLVTLFK,a Selective Agonist of Nonopioid β-Endorphin Receptor,Reduces the Corticotropin and Corticosterone Response

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β-endorphin, a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of the specific binding of [³H]octarphin to anterior pituitary membranes obtained from rats before and after the lipopolysaccharide (LPS)-injection showed that 2 h after LPS administration the value of maximal binding capacity of the membranes (B_max) was increased by 1.6 times (B_max 12.3 ± 0.8 and 20.0 ± 1.9 pmol/mg of protein, respectively), while the binding affinity was not changed (K _d 5.8 ± 0.3 and 5.5 ± 0.4 nM, respectively). At the same time, LPS did not have a significant effect on the characteristics of the labeled peptide binding to adrenal cortex membranes. Intranasal injection of octarphin at doses of 10–30 μg/rat was found to reduce the LPS-induced corticotropin and corticosterone response. The effect of the peptide was dose-dependent with a maximum at a dose 20 μg/rat. Aminoguanidine (AG 100 mg/kg i.p.), a selective inducible nitric oxide synthase (iNOS) inhibitor, completely abolished the inhibitory effect of the peptide on the LPS-induced corticotropin and corticosterone response. At the same time, octarphin in vitro stimulated in a time- and concentration-dependent manner the anterior pituitary iNOS expression of rats injected with LPS (1 mg/kg i.p.). The maximum level of the iNOS expression was observed at a peptide concentration of 10 nM after 2 h cultivation. These results indicate that the inhibitory effect of octarphin on LPS-induced secretion of corticotropin and corticosterone due to the ability of the peptide to stimulate the expression of iNOS in the anterior pituitary.     

Binding of β-endorphin and its fragments to the nonopiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid β-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 β-endorphin receptor of mouse peritoneal macrophages (K _d 2.4 ± 0.1 nM). The specific binding of [³H]immunorphin to macrophages was inhibited by unlabeled β-endorphin (K _i of the [³H]immunorphin-receptor complex 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met⁵]enkephalin (K _i > 10 μM). Thirty fragments of β-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 β-endorphin is its fragment 12–19 (K _i 3.1 ± 0.3 nM).     

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

-Endorphin and the synthetic -endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met⁵]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of -endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of ¹²⁵I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K _d = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits ¹²⁵I-labeled -endorphin specific binding to naloxone insensitive receptors on T lymphocytes (K _i = 0.6 ± 0.1 nM)). Thus, -endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.     

An ACTH-Like Peptide Immunocortin: Effect on the Activity of Cells from the Rat Adrenal Cortex

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 g/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 g/kg exerted practically no effect on the CS content and its dose of 1000 g/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 K _i of 1.2 nM).     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

Molecular phylogeny of fungus gnats (Diptera: Mycetophilidae) revisited: position of Manotinae,Metanepsiini, and other enigmatic taxa as inferred from multigene analysis

The phylogeny of selected genera from four subfamilies of fungus gnats (Diptera: Mycetophilidae) – Manotinae, Leiinae, Sciophilinae and Gnoristinae (including Metanepsiini) – is reconstructed based on the combined analysis of five mitochondrial (12S, 16S, COI, COII, cytB) and two nuclear (28S, ITS2) gene markers. Results of the different analyses all support Manotinae as a monophyletic group, with Leiinae as the sister group. Allactoneura DeMeijere is nested in the monophyletic and strongly supported clade of Leiinae. The tribe Metanepsiini is revealed as paraphyletic and the genera Metanepsia Edwards and Chalastonepsia Søli do not appear to be closely related. The genera Docosia Winnertz, Ectrepesthoneura Enderlein, Novakia Strobl and Syntemna Winnertz were placed with a group of genera included traditionally in the Gnoristinae. The monophyly of Dziedzickia Johannsen and Phthinia Winnertz is not supported. The genera of Sciophilinae (excluding Paratinia Mik but including Eudicrana Loew) form a monophyletic group in the Bayesian model.     

A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.