FMRFamide-related peptides (FaRPs): A new family of peptides from amphibian defensive skin secretions

Amphibian defensive skin secretions are known to contain a plethora of biologically-active peptides that are often structural and functional analogues of vertebrate neuropeptides. Here we report the structures of two invertebrate neuropeptide analogues, IPPQFMRF amide (IF-8 amide) and EGDEDEFLRF amide (EF-10 amide), from the defensive skin secretions of two different species of African hyperoliid frogs, Kassina maculata and Phylictimantis verrucosus, respectively. These represent the first canonical FMRF amide-related peptides (FaRPs) from a vertebrate source. The cDNA encoding IF-8 amide was cloned from a skin secretion library and found to contain a single copy of the peptide located at the C-terminus of a 58 amino acid residue open-reading frame. These data extend the potential targets of the defensive arsenal of amphibian tegumental secretions to parasitic/predatory invertebrates and the novel peptides described may represent the first vertebrate peptidic endectocides.

Sauvatide - a novel amidated myotropic decapeptide from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei.

Here we describe the structural and functional characterization of a novel myotropic peptide, sauvatide, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. Sauvatide is a C-terminally amidated decapeptide with the following primary structure – LRPAILVRTKamide – monoisotopic mass 1164.77 Da, which was found to contract the smooth muscle of rat urinary bladder with an EC50 of 2.2 nM. The sauvatide precursor, deduced from cloned skin cDNA, consists of 62 amino acid residues with a single copy of sauvatide located near the C-terminus. The mature peptide is generated from the precursor by cleavage at a classical –KR-cleavage site located proximal to the N-terminus and by removal of a –GKGK sequence at the C-terminus, the first glycyl residue acting as amide donor. Amphibian skin secretions thus continue to be a source of novel and potent biologically active peptides acting through functional targets in mammalian tissues.

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide: oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

The redox-Bohr group associated with iron-sulfur cluster N2 of complex I

Proton pumping respiratory complex I (NADH: ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

Risk factors for the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection in 59 Irish dairy herds

Since 1994, Irish cattle have been exposed to greater risks of acquiring Mycobacterium avium subspecies paratuberculosis (MAP) infection as a consequence of the importation of over 70,000 animals from continental Europe. In recent years, there has been an increase in the number of reported clinical cases of paratuberculosis in Ireland. This study examines the prevalence of factors that promote the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) on selected Irish dairy farms in the Cork region, and the association between these factors and the results of MAP screening tests on milk sock filter residue (MFR). A total of 59 dairy farms, selected using non-random methods but apparently free of endemic paratuberculosis, were enrolled into the study. A questionnaire was used to collect data about risk factors for MAP introduction and transmission. The MFR was assessed on six occasions over 24 months for the presence of MAP, using culture and immunomagnetic separation prior to polymerase chain reaction (IMS-PCR). Furthermore, blood samples from all entire male and female animals over one year of age in 20 herds were tested by ELISA. Eighteen (31%) farms had operated as closed herds since 1994, 28 (47%) had purchased from multiple sources and 14 (24%) had either direct or indirect (progeny) contact with imported animals. Milk and colostrum were mixed on 51% of farms, while 88% of farms fed pooled milk. Thirty (51%) herds tested negative to MFR culture and IMS-PCR, 12 (20%) were MFR culture positive, 26 (44%) were IMS-PCR positive and seven (12%) were both culture and IMS-PCR positive. The probability of a positive MFR culture was significantly associated with reduced attendance at calving, and with increased use of individual calf pens and increased (but not significantly) if multiple suckling was practised. There was poor agreement between MFR culture and MFR IMS-PCR results, but moderate agreement between MFR culture and ELISA test results. This study highlights a lack of awareness among Irish dairy farmers about the effect of inadequate biosecurity on MAP introduction. Furthermore, within-herd transmission will be facilitated by traditional calf rearing and waste management practices. The findings of viable MAP in the presence of known transmission factors in non-clinically affected herds could be a prelude to long-term problems for the Irish cattle and agri-business generally.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

Phylloseptin-1 (PSN-1) from Phyllomedusa sauvagei skin secretion: a novel broad-spectrum antimicrobial peptide with antibiofilm activity

Amphibian skin secretions have proven to be rich sources of antimicrobial peptides that are proposed to be fundamental components of the innate immune system. As amphibian skin is a multi-functional organ playing, among other things, a crucial role in respiration, it has been deemed that a core biological role for such peptides is control of microbial flora on this surface. To date, however, antimicrobial efficacy has been universally determined by means of establishing minimum inhibitory concentrations (MICs) using planktonic organisms rather than those within a biofilm such as would occur on this exposed surface. Here we describe the identification and structural characterisation of a novel 19 amino acid residue antimicrobial peptide of the phylloseptin family, named PSN-1, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. PSN-1 displayed broad-spectrum activity against a range of planktonic organisms with a high potency (MIC 5 µM) against Staphylococcus aureus. In a specific bioassay with the same organism grown as a biofilm, the minimal biofilm eradication concentration (MBEC) was found to be of the same high potency (5 µM). The present data would suggest that evaluation of actions and potency of amphibian skin secretion antimicrobial peptides might best be achieved by evaluating MBEC rather than MIC using planktonic organisms and that data arising from such studies may have more biological relevance in reflecting the purpose for which they have evolved through natural selection.

The structure of helokinestatin-5 and its biosynthetic precursor from Gila monster (Heloderma suspectum) venom: Evidence for helokinestatin antagonism of bradykinin-induced relaxation of rat tail artery smooth muscle

Here we report the primary structure of a novel peptide, named helokinestatin-5 (VPPPLQMPLIPR), from the venom of the Gila monster (Heloderma suspectum). Helokinestatin-5 differs in structure from helokinestatin-3 by deletion of a single prolyl residue in the N-terminally located polyproline region. Two different biosynthetic precursors were consistently cloned from a venom-derived cDNA library. The first encoded helokinestatins 1–4 and a single copy of C-type natriuretic peptide, as previously described, whereas the second was virtually identical, lacking only a single prolyl codon as found in the mature attenuated helokinestatin-5 peptide. Helokinestatins 1–3 and 5 were synthesized by solid-phase fmoc chemistry and each synthetic replicate was found to antagonize the relaxation effect induced by bradykinin on rat tail artery smooth muscle. Helokinestatins thus represent a novel family of vasoactive peptides from the venom of helodermatid lizards

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Hylambatin and (Thr)11-hylambatin from the skin secretion of the African hyperoliid frog, Kassina maculata: Structural characterisation, precursor cDNA cloning and preliminary pharmacological testing

The tachykinins hylambatin and (Thr)11-hylambatin have been isolated from the defensive skin secretion of the African hyperoliid frog, Kassina maculata,. Hylambatin (DPPDPNRFYGMMamide) is revised in structure from the original sequence by a single site substitution (Asn/Asp at position 6), and (Thr)11-hylambatin, a novel tachykinin, differs in structure from hylambatin by a single Thr/Met substitution. (Thr)11-hylambatin is five- to ten-fold more abundant than hylambatin in secretions. Synthetic replicates of both peptides were active in smooth muscle preparations including the rat tail artery, rat ileum and bovine trachea. While hylambatin displayed activity consistent with an NK1-receptor ligand, (Thr)11-hylambatin was more active than either substance P or neurokinin A in both NK1- and NK-2 receptor rich preparations. Incorporation of a threoninyl residue rather than the canonical leucyl residue at the penultimate position in both substance P and neurokinin A, generated active ligands in both arterial and intestinal smooth muscle preparations. Hylambatin precursor cDNAs, designated HYBN-1 and HYBN-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. Both were highly-homologous containing open-reading frames of 66 amino acids encoding single copies of either hylambatin or (Thr)11-hylambatin. These data reveal a hitherto unrecognized structure/activity attribute of mammalian tachykinin receptors revealed though discovery of a novel amphibian skin-derived, site-substituted peptide ligand.

GK-22 amide: a novel myotropic peptide from the skin secretion of the bamboo leaf odorous frog, Odorrana versabilis

The skin secretions produced by many amphibians are formidable chemical/biological weapons deployed as a defence against predators. Bioactive peptides are often the predominant class of biochemical within these secretions and the inventory of such remains incomplete with each individual taxon producing unique cocktails contained within which are some signature peptides, such as bradykinins and tachykinins. These secretions have been the source of many peptides subsequently found to have structural homologues in vertebrate neuroendocrine systems (bombesin/GRP; sauvagine/CRF; caerulein/CCK) and vice versa (bradykinin, CGRP, NMU). They are thus unequivocally intriguing resources for novel bioactive peptide discovery. Here we describe a novel 22-mer amidated peptide, named GK-22 amide (N-terminal Gly (G) and C-terminal Lys (K) amide) with an internal disulphide bridge between Cys (C) 11 and 20 from the skin secretion of Odorrana versabilis. Molecular cloning indicated that it is encoded as a single copy on a biosynthetic precursor of 59 amino acid residues consisting of a signal peptide, an acidic amino acid residue-rich spacer domain and a mature peptide encoding domain flanked N-terminally by a classical -Lys-Arg- (KR) propeptide convertase processing site and C-terminally by a Gly (G) residue amide donor. A synthetic replicate of this peptide produced potent and dose-dependent contraction of the smooth muscle of rat urinary bladder. GK-22 amide thus represents the prototype of a novel class of myotropic peptide from amphibian skin and its discovery illustrates the continuing potential of this resource to this end.

Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.

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.

Effect of growth-promoting 17ß estradiol, 19 nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves

The use of screening methods based on the detection of biological effects of growth promoters is a promising approach to assist residue monitoring. To reveal useful effects on protein metabolism, male and female veal calves at 10 weeks of age were treated thrice with a combination of 25 mg 17ß-estradiol 3-benzoate and 150 mg 19-nortestosterone decanoate with 2 weeks intervals and finally once with 4 mg dexamethasone. Hormone-treated calves showed a significant accelerated growth rate over 6 weeks. Plasma samples of treated and control calves were analysed for immunoreactive inhibin (ir-inhibin), osteocalcin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), IGFBP-3, luteinzing hormone (LH), follicle-stimulating hormone (FSH) and prolactin using immunoaffinity assays. Hormone treatment did not affect levels of IGF-1, IGFBP-2, IGFBP-3, LH, FSH and prolactin. The concentration of circulating ir-inhibin decreased, however, significantly (P < 0.05) in bull calves upon administration of the sex steroids, whereas it remained unchanged in the female animals. Dexamethasone treatment decreased significantly (P < 0.05) circulating levels of osteocalcin in both female and male animals. Ir-inhibin and osteocalcin were, therefore, considered as candidates for a protein biomarker-based screening assay for detection of abuse of estrogens, androgens and/or glucocorticoids in cattle fattening, which is being developed in the framework of EU research project BioCop

Evidence of natural occurrence of the banned antibiotic chloramphenicol in herbs and grass

Chloramphenicol (CAP), a broad-spectrum antibiotic, was detected in several herb and grass samples from different geographic origins. Due to its suspected carcino-genicity and linkages with the development of aplastic anemia in humans, CAP is banned for use in food-producing animals in the European Union (EU) and many other countries. However, products of animal origin originating from Asian countries entering the European market are still found noncompliant (containing CAP) on a regular basis, even when there is no history of chloramphenicol use in these countries. A possible explanation for the continued detection of these residues is the natural occurrence of CAP in plant material which is used as animal feed, with the consequent transfer of the substance to the animal tissues. Approximately 110 samples were analyzed using liquid chromatography coupled with mass spectrometric detection. In 26 samples, the presence of CAP was confirmed using the criteria for banned substances defined by the EU. Among other plant materials, samples of the Artemisia family retrieved from Mongolia and from Utah, USA, and a therapeutic herb mixture obtained from local stores in the Netherlands proved to contain CAP at levels ranging from 0.1 to 450 mu g/kg. These findings may have a major impact in relation to international trade and safety to the consumer. The results of this study demonstrate that noncompliant findings in animal-derived food products may in part be due to the natural occurrence of chloramphenicol in plant material. This has implications for the application of current EU, USA, and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.