Identification and molecular cloning of novel trypsin inhibitors from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata).

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.

A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.

Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis.

In this study, an amphibian (Odorrana hejiangensis) skin extract was fractionated by reverse phase HPLC and fractions were screened for trypsin inhibitory activity. Using this initial approach, a novel trypsin inhibitory peptide was detected with an apparent protonated molecular mass of 1804.83Da, as determined by MALDI-TOF mass spectrometry. It was named Hejiang trypsin inhibitor (HJTI) in accordance. The primary structure of the biosynthetic precursor of HJTI was deduced from a cDNA sequence cloned from a skin-derived cDNA library. The primary structure of the encoded predicted mature active peptide was established as: GAPKGCWTKSYPPQPCS (non-protonated monoisotopic molecular mass - 1802.81Da). On the basis of this unequivocal amino acid sequence, a synthetic replicate was synthesized by solid phase Fmoc chemistry. This replicate displayed a moderately potent trypsin inhibition with a K(i) of 388nM. Bioinformatic analysis of the primary structure of this peptide indicated that it was a member of the Bowman-Birk family of protease inhibitors. The substitutions of Gln-14 and Ser-17 by Lys, resulted in an increase in cationicity and a small increase in potency to a K(i) value of 218nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity.

THE ULTRASTRUCTURE AND IMMUNOGOLD LABELING OF PANCREATIC POLYPEPTIDE-IMMUNOREACTIVE CELLS ASSOCIATED WITH THE EGG-FORMING APPARATUS OF A MONOGENEAN PARASITE, DICLIDOPHORA-MERLANGI

An electron microscopical examination has been made of the fine structure and disposition of pancreatic polypeptide immunoreactive cells associated with the egg-forming apparatus in Diclidophora merlangi. The cell bodies are positioned in the parenchyma surrounding the ootype and taper to axon-like processes that extend to the ootype wall. The terminal regions of these processes branch and anastomose and, in places, the swollen endings or varicosities form synaptic appositions with the muscle fibres in the ootype wall. The cells are characterized by an extensive GER-Golgi system that is involved in the assembly and packaging of dense-cored vesicles. The vesicles accumulate in the axons and terminal varicosities, and their contents were found to be immunoreactive with antisera raised to the C-terminal hexapeptide amide of pancreatic polypeptide. It is concluded that the cells are neurosecretory in appearance and that, functionally, their secretions may serve to regulate ootype motility and thereby help co-ordinate egg production in the worm.

THE ORGANIZATION OF THE NERVOUS-SYSTEM IN PLATHELMINTHES - THE NEUROPEPTIDE F-IMMUNOREACTIVE PATTERN IN CATENULIDA, MACROSTOMIDA, PROSERIATA

The organization of the nervous system of Archilopsis unipunctata Promonotus schultzei and Paramonotus hamatus (Monocelididae, Proseriata) and Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) was studied by immunocytochemistry, using antibodies to the authentic flatworm neuropeptide F (NPF) (Moniezia expansa). The organization of the nervous system of the Monocelididae was compared to that of the nervous system of Bothriomolus balticus (Otoplanidae), a previously studied species of another family of the Proseriata. The results show that the main nerve cords (MCs), independent of lateral or ventral position in the Monocelididae and the Otoplanidae, correspond to each other. The study also confirms the status of the lateral cords as main cords (MCs) in S. leucops and M. lineare. Common for MCs in the members of the investigated taxa are the following features: MCs consist of many fibres, originate from the brain and are adjoined to 5-HT-positive neurons. In Monocelididae and Otoplanidae, the MCs additionally have the same type of contact to the pharyngeal nervous system. Also common for both proseriate families is the organization of the two lateral nerve cords, with weaker connections to the brain, and the pair of dorsal cords running above the brain. The organization of the minor cords differs. The Monocelididae have a pair of thin ventral cords forming a mirror image of the dorsal pair. Furthermore, an unpaired ventral medial cord connecting medial commissural cells was observed in P. schultzei. Marginal nerve cords, observed in Otoplanidae, are absent in Monocelididae. All minor nerve cords are closely connected to the peripheral nerve plexus. The postulated trends of condensation of plexal fibres to cords and/or the flexibility of the peripheral nerve plexus are discussed. In addition, the immunoreactivity (IR) pattern of NPF was compared to the IR patterns of the neuropeptide RFamide and the indoleamine, 5-HT (serotonin). Significant differences between the distribution of IR to NPF and to 5-HT occur. 5-HT-IR dominates in the submuscular and subepidermal plexuses. In the stomatogastric plexus of M. lineare, only peptidergic IR is observed in the intestinal nerve net. The distribution of NPF-IR in fibres and cells of the intestinal wall in M. lineare indicates a regulatory function for this peptide in the gut, while a relationship with ciliary and muscular locomotion is suggested for the 5-HT-IR occurring in the subepidermal and submuscular nerve plexuses. In M. lineare, the study revealed an NPF- and RFamide-positive cell pair, marking the finished development of new zooids. This finding indicates that constancy of these cells is maintained in this asexually reproducing and regenerating species.

LOCALIZATION, QUANTITATION, AND CHARACTERIZATION OF NEUROPEPTIDE-F-IMMUNOREACTIVE AND FMRFAMIDE-IMMUNOREACTIVE PEPTIDES IN TURBELLARIANS AND A MONOGENEAN - A COMPARATIVE-STUDY

Over the past decade it has become clear that the nervous systems of platyhelminths are both complex and highly developed, particularly in peptidergic elements. The central position of an ancestral flatworm in the evolution of the Bilateria has placed a greater importance on the study of modern flatworms. Using antisera generated to the C-terminal region of platyhelminth neuropeptide F and the molluscan neuropeptide, FMRFamide, in immunocytochemistry at both Light and ultrastructural levels, immunoreactivities have been localised within the nervous systems of three species of triclad turbellarians, Dugesia lugubris, Dendrocoelum lacteum, and Polycelis nigra, and one species of monogenean trematode, Diclidophora merlangi. Extensive immunostaining was obtained with both antisera throughout the central and peripheral nervous systems of all species studied, but intensity and abundance was significantly greater in the turbellarians. Indirect electron-immunogold labeling demonstrated that immunoreactivity to both neuropeptides was often colocalised in neurosecretory vesicles, although discrete populations of vesicles were also observed. Radioimmunoassay of extracts of all species confirmed that neuropeptide F immunoreactivity was consistently more abundant than FMRFamide immunoreactivity, and that the levels of both in the three turbellarians were several orders of magnitude greater than those found in the monogenean. Chromatographic analyses of turbellarian extracts revealed that neuropeptide F and FMRFamide immunoreactivities were attributable to different peptides. These data imply that the neuropeptidergic systems of turbellarians are considerably more extensive than those of monogeneans, and would suggest that a regression has occurred in the latter as a consequence of the adoption of a more sedentary parasitic lifestyle. (C) 1995 Wiley-Liss, Inc.