Identification of a new myotropic decapeptide from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas

Bradykinin-related peptides (BRPs) family is one of the most significant myotropic peptide families derived from frog skin secretions. Here, a novel BRP callitide was isolated and identified from the red-eyed leaf frog, Agalychnis callidryas, with atypical primary structure FRPAILVRPK-NH2. The mature peptide was cleaved N-terminally at a classic propeptide convertase cleavage site (-KR-) and at the C-terminus an unusual -GKGKGK sequence was removed using the first G residue as an amide donor for the C-terminally-located K residue. Thereafter, the synthetic replicates of callitide were assessed the myotropic activity and showed a significant contraction of balder, with the 0.63 nM EC50 value, more potent than most discovered myotropic peptides. The binding mode was further speculated by molecular docking and stimulation. The result indicated that the C-terminal of callitide might selectively bind to bradykinin receptor B2 (BKRB2). Further investigation of the callitide needs to be done in the future to be exploited as potential future drug leads.

Sulfakinin Signalling Influences Fatty Acid Levels and Composition in Tenebrio Molitor Beetle

Background: Sulfakinins are arthropod neuropeptides that are structurally and functionally similar to vertebrate gastrin-cholecystokinin. Sulfakinins with sulfated tyrosine (sSK) or nonsulfated tyrosine (nSK) in the C-terminated heptapeptide XY(SO3H)GHMRFamide display different biological functions, including myotropic activity, inhibition of food intake, stimulation of digestive enzymes and regulation of carbohydrate and lipid content. Objective: To reveal the mechanisms by which sulfakinin signalling modulates lipid homeostasis, we analysed the changes in the level and composition of fatty acids and organic compounds in the fat body and haemolymph of Tenebrio molitor larvae after nSK and sSK treatment. Methods: Fatty acids in fat body and haemolymph of insects were analysed using Gas Chromatography - Mass Spectrometry (GC–MS). Results: The direction of the changes observed for major fatty acids, 18:1 and 18:2, and the less abundant fatty acids, 16:0, 18:0, 16:1 and 14:0, was the same for unsaturated (UFAs) and saturated (SFAs) fatty acids, and elevated after nSK application in both analysed tissues. However, the action of sSK in fat body tissue evoked distinct effects and induced either significant decreases in individual fatty acids or UFAs and SFAs. Administration of nSK and sSK significantly increased the level of total organic compounds in the haemolymph, contrary to the effect of sSK in fat body, where the level of total organic compounds decreased, although changes differ between individual chemicals. Conclusion: Sulfakinins are engaged in the precise modulation of fatty acid levels and composition, but their action depends on the presence of sulfate group on the tyrosyl residue of the peptide what determines the different roles of these peptides in insect physiology.

Biological evaluation of analogues of an insect neuropeptide proctolin.

Continuing our studies on proctolin (Arg-Tyr-Leu-Pro-Thr) we performed the synthesis and biological evaluation of 52 analogues substituted in position 2, 3, 4, and 5 of the peptide chain. The peptides were bioassayed for cardiotropic activity in vitro on Tenebrio molitor and myotropic activity on foregut of Schistocerca gregaria. Twenty analogues retained 20-80% of proctolin activity.

Gastrin-releasing peptides from Xenopus laevis: purification, characterization, and myotropic activity

Two molecular forms of gastrin-releasing peptide (GRP) were isolated from an extract of the intestine of the tetraploid frog Xenopus laevis. The primary structure of GRP-1 (APTSQQHTEQ10LSRSNINTRG20 SHWAVGHLM.NH2) differs from that of GRP-2 by a single amino acid substitution (Asn15→ Thr15). GRP-(20–29) peptide (neuromedin C) was also isolated from the extract. Synthetic GRP-1 produced concentration-dependent contractions of longitudinal smooth muscle strips from Xenopus cardiac stomach (pD2 = 8.93 ± 0.32; n = 6). The responses were unaffected by tetrodotoxin, atropine, and methysergide, indicating a direct action of the peptide on smooth muscle cells. GRP-1 elicited concentration-dependent relaxations of precontracted (5 μM carbachol) circular smooth muscle strips from the same region (pD2 = 8.96 ± 0.21; n = 8). The responses were significantly ( P < 0.05) attenuated (71 ± 24% decrease in maximum response; n = 6) by indomethacin, indicating mediation, at least in part, by prostanoids. Despite the fact that Xenopus GRP-1 differs from pig GRP at 15 amino acid sites, both peptides are equipotent and equally effective for both contractile and relaxant responses, demonstrating that selective evolutionary pressure has acted to conserve the functional COOH-terminal domain in the peptide. The data suggest a physiologically important role for GRP in the regulation of gastric motility in X. laevis.

Purification, structural characterization, and myotropic activity of endothelin from trout, Oncorhynchus mykiss

Endothelin (ET) from a nontetrapod species has never been characterized, either structurally or biologically. A single molecular form of trout ET with 21-amino-acid residues was isolated in pure form from an extract of the kidney of the steelhead trout, Oncorhynchus mykissand its primary structure established as Cys-Ser-Cys-Ala-Thr-Phe-Leu-Asp-Lys-Glu10-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile20-Trp. This amino acid sequence shows only three substitutions (Ala4→Ser, Thr5→Ser, and Phe6→Trp) compared with human ET-2, demonstrating that the structure of the peptide has been well conserved during evolution and that the pathway of posttranslational processing of preproendothelin in the trout is probably similar to that in mammals. Synthetic trout ET produced concentration-dependent constrictions of isolated rings of vascular tissue from trout efferent branchial artery (EBA; pD2 = 7.90 ± 0.06, n = 5), caeliacomesenteric artery (pD2 = 8.03 ± 0.04, n = 4), anterior cardinal vein (ACV; pD2 = 8.57 ± 0.25, n = 4), and rat abdominal aorta (AO; pD2 = 8.86 ± 0.08, n = 7). Trout and rat vessels were more sensitive to mammalian ET-1 than to trout ET (pD2 for human ET-1 in: EBA = 9.12 ± 0.14; ACV = 9.90 ± 0.15; AO = 8.86 ± 0.08), but there was no significant difference in the maximum tension produced by either peptide in these vessels.