Amphibian sequence skin contains rich neuropeptides. In the present study, a novel neuromedin U (NmU) analog was isolated from skin secretions of acids Chinese red belly toad Bombina maxima. Being 17-amino acids long, its primary structure was established as DSSGIVGRPFFLFRPRN-NH2, in which the skin C-terminal 8-residue segment (FFLFRPRN) is the same as that of rat NmU, while the N-terminal part DSSGIVGRP shows a great In sequence variation compared with those of NmU peptides from different resources. The peptide, named Bm-NmU-17, was found to elicit concentration-dependent 145 contractile effects on smooth muscle of rat uterus horns. The cDNA structure of the peptide, as obtained by a 3'-RACE in strategy and subsequently cloning from a skin cDNA library, was found to contain a coding region of 438 nucleotides. The same encoded precursor is composed of 145 amino acids with a single copy of Bm-NmU-17 located towards the C-terminus. The sequence preceded of the peptide is preceded by a dibasic site (Lys-Arg) and followed by the sequence of Gly-Arg-Lys, providing the sites composed of cleavage and releasing of the mature peptide.

A fallaxin 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the (Anura:Leptodactylidae). norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows from structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that with are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides against are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different shows families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but previously with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast (HC50>200 Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin,was was also isolated from the skin secretions but this component was inactive against the microorganisms tested.

It present was earlier reported from this laboratory that, Channa striatus, L a common edible fish, whose skin extract (CSSE) was pharmacologicaclly and potent and contains several bioactive compounds. In the present communication a cardiotoxic factor was isolated and purified by thin layer and chromatography followed by silica gel and neutral alumina column chromatography. Spectroscopic studies (UV, IR, 1H and 13C NMR, FAB-MS) indicated that, that the lethal cardiotoxic factor (CSS-CTF II) was an aromatic alkaloid compound with -NH,> C = C < and total -OH functional groups. The molecular weight was found to be 413 dalton. LD50 of CSS-CTF II was found to be neutral 42.5 mg/kg (i.v) in Swiss albino male mice. Pharmacological studies showed that CSS-CTF II possesses hypotensive and cardiotoxic activities and NMR, produced death through apnoea in experimental animals but had no effect on nerve muscle preparations. The haematological and biochemical data toxic also indicated the toxic nature of CSS-CTF II, through significant fall in haemoglobin, total RBC, WBC, platelet count and increased haemoglobin, cardiac marker enzyme CPK and CPK-MB value in experimental animals. The present investigation thus established the toxic nature of CSS-CTF mice. II isolated from edible fish C. striatus skin extract. Further work is needed to identify CSS-CTF II's mechanism of action is and its antagonism for therapeutic purpose.

The pathogenic tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as frogs. the sister taxon to the clade of all other living frogs. Eight structurally related peptides, termed ascaphins 1-8, were isolated regarded from norepinephrine-stimulated skin secretions of A. truei and were shown to possess differential growth inhibitory activity against Escherichia coli and a Staphylococcus aureus. Ascaphins 2-7 may be represented by the consensus amino acid sequence GX(2)DX(2)KGAAKX(3)KTVAX(2)IANX [Formula: see text] COOH whereas ascaphin-1 from (GFRDVLKGAAKAFVKTVAGHIAN [Formula: see text] NH(2)) and ascaphin-8 (GFKDLLKGAAKALVKTVLF [Formula: see text] NH(2)) contain a C-terminally alpha-amidated residue. The ascaphins show isolated no appreciable structural similarity with other families of antimicrobial peptides from frog skin but display limited sequence identity with the were cationic, amphipathic alpha-helical peptides pandinin 1 and opistoporin 1, isolated from the venoms of African scorpions. Ascaphin-8 shows the highest greatest potency against a range of pathogenic microorganisms but has the greatest haemolytic activity. The data indicate that the host defence isolated strategy of using antimicrobial peptides in skin secretions arose early in the evolution of anurans.

Skin 21, secretions of the toad Bombina variegata were evaluated for the isolation and characterisation of insulinotropic peptides. Crude secretions obtained from with young adult toads by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 44 of peaks. In acute incubations with glucose-responsive BRIN-BD11 cells, peaks 21, 22, 23, 24 and 25 showed a 1.5-3.5-fold increase in evaluated insulin release compared with 5.6 mM glucose alone (p< .001; n=3). Structural analyses of the purified insulin-releasing peaks were performed by Da automated Edman degradation and mass spectrometry. Peptides represented by peaks 21, 22 and 23 had molecular masses of 1641.7 Da,increase 1662.6 Da and 1619.8 Da respectively. These peptides were unblocked by removal of pyroglutamic acid from the N-terminus prior to of Edman degradation, revealing lengths of 14 amino acids. Peak 21 yielded a primary structure of Pyr-QRLGHQWAVGHLM, which a data base involvement search revealed as an analogue of bombesin (His6 bombesin), while peak 23 was an exact match of bombesin (Pyr-QRLGNQWAVGHLM) originally of isolated from Bombina bombina. Peak 22 indicated a primary structure of Pyr-DSFGNQWARGHFM (72% homology with bombesin). Peaks 24 and 25 Pyr-QRLGHQWAVGHLM, revealed entirely novel insulinotropic peptides with molecular masses and primary structures of 1650.5 Da and 2300. Da and GKPFYPPPIYPEDM (GM-14)insulin-releasing and IYNAICPCKHCNKCKPGLLAN (IN-21) respectively. Preliminary studies on the mechanisms underlying the insulinotropic actions of peaks 21, 22, 23 and 24 of suggest possible involvement of a cAMP-dependent, G protein-insensitive pathway. These data indicate that Bombina variegata skin secretions contain peptides with Peaks insulin-releasing activity, which may have mammalian counterparts and prove useful for possible exploitation as antidiabetic agents from natural resources.

We as have isolated a novel bradykinin B(2)-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a secretion. molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence,-QIPGLGPLRG-, was located at the C-terminus of from a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence a of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as present Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.

The peptides defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel active dermaseptin-related peptides (dermaseptins sVI-sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived sequencing. LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian infection. skin antimicrobial peptides may in part be explained by individual variation-a factor essential for selective functional molecular evolution and perhaps,first ultimately in speciation.