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.

Construction of cDNA libraries from trifluoroacetic acid-solvated amphibian skin secretions: molecular cloning of multiple bombinin-like peptide precursor transcripts from a library of yellow-bellied toad (Bombina variegata) secretion

Amphibian skin secretions are renowned as complex mixtures of bioactive peptides many of which are analogues of endogenous regulatory peptides. While skin secretions can be obtained non-invasively for peptidome analysis, parallel studies on the granular gland transcriptome required specimen sacrifice. The aim of the present study was to analyse archived skin secretions to determine the robustness of bioactive peptide precursor-encoding polyadenylated mRNAs in an attempt to extract maximum molecular information from rare samples. A range of solvated skin secretion samples were examined after lyophilisation for their potential to generate viable and comprehensive cDNA libraries based upon polyadenylated mRNA capture and amplification/cloning using appropriate commercial kits. Here we present unequivocal data that the granular gland transcriptome persists in a PCR amenable format even after storage for as long as 12 years in 0.1%(v/v) aqueous trifluoroacetic acid (TFA). We used a pooled skin secretion sample (2 ml) from the yellow-bellied toad, Bombina variegata (n = 14), containing the equivalent of 5 mg/ml of lyophilised skin secretion, that had been used in part for peptide isolation purposes in 1998 and had been stored at - 20 °C since that time. In the first cloning experiment, 12 different bombinin-like peptide precursor cDNAs were cloned encoding 17 different bombinins, the majority of which were novel. Subsequently, bombesin and bradykinin-related peptide precursor transcripts have been cloned successfully. These data illustrate the unexpected stability/longevity of the transcriptome in these secretions — a finding with implications for both this field of research and for the wider field of molecular biology.

The carbenoid approach to peptide synthesis

A different approach to the synthesis of dipeptides is described based on the formation of the (NHCHRCONH)-C-1-(CHRCO)-C-2 bond by carbenoid N-H insertion, rather than the formation of the peptide bond itself. Thus decomposition of triethyl diazophosphonoacetate catalysed by rhodium(Ii) acetate in the presence of N-protected amino acid amides 8 gives the phosphonates 9, Subsequent Wadsworth-Emmons reaction of 9 with aldehydes in the presence of DBU gives dehydro dipeptides 10. The reaction has been extended to a simple two-step procedure, without the isolation of the intermediate phosphonate. for conversion of a range of amino acid amides 11 into dehydro dipepides 12 and to an N-methylamide 11h, and for conversion of a dipeptide: to tripeptide (13-14). Direct conversion, by using methyl diazophenylacetate, of amino acid amides to phenylglycine-containing dipeptides 19 proceeds in good chemical yield, but with poor diastereoselectivity.

A new approach to peptide synthesis

A new approach to the synthesis of dipeptides is described based on the formation of the CONH-CHRCO bond by carbenoid N-H insertion, rather than the formation of the peptide bond itself. The key N-H insertion reaction was carried out by treating a mixt. of N-protected amino acid amide and tri-Et diazophosphonoacetate, EtO2CC(:N2)PO(OEt)2, with a catalytic amt. of Rh2(OAc)4 in toluene to form phosphonates, e.g. I (R1 = H, Me, iso-Pr, iso-Bu; R2 = PhCH2O2C, Me3CO2C) in good yield. Dehydro dipeptides, e.g. II (R1, R2 = same as above; R3 = Ph, iso-Pr, N-Boc-indol-3-yl) were prepd. by Wadsworth-Emmons reaction of the phosphonates I with R3CHO using DBU as base.

Intermedin (Adrenomedullin-2) a novel counter-regulatory peptide in the cardiovascular and renal systems

Intermedin (IMD) is a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). Proteolytic processing of a larger precursor yields a series of biologically active C-terminal fragments, IMD1–53, IMD1–47 and IMD8–47. IMD shares a family of receptors with AM and CGRP composed of a calcitonin-receptor like receptor (CALCRL) associated with one of three receptor activity modifying proteins (RAMP). Compared to CGRP, IMD is less potent at CGRP1 receptors but more potent at AM1 receptors and AM2 receptors; compared to AM, IMD is more potent at CGRP1 receptors but less potent at AM1 and AM2 receptors. The cellular and tissue distribution of IMD overlaps in some aspects with that of CGRP and AM but is distinct from both. IMD is present in neonatal but absent or expressed sparsely, in adult heart and vasculature and present at low levels in plasma. The prominent localization of IMD in hypothalamus and pituitary and in kidney is consistent with a physiological role in the central and peripheral regulation of the circulation and water-electrolyte homeostasis. IMD is a potent systemic and pulmonary vasodilator, influences regional blood flow and augments cardiac contractility. IMD protects myocardium from the deleterious effects of oxidative stress associated with ischaemia-reperfusion injury and exerts an anti-growth effect directly on cardiomyocytes to oppose the influence of hypertrophic stimuli. The robust increase in expression of the peptide in hypertrophied and ischaemic myocardium indicates an important protective role for IMD as an endogenous counter-regulatory peptide in the heart.

Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

The inhibitor profiling of the caspase family of proteases using substrate-derived peptide glyoxals.

A series of substrate-based a-keto-ß-aldehyde (glyoxal) sequences have been synthesised and evaluated as inhibitors of the caspase family of cysteine proteases. A number of potent inhibitor sequences have been identified. For example, a palmitic acid containing sequence pal-Tyr-Val-Ala-Asp-glyoxal was demonstrated to be an extremely effective inhibitor of caspase-1, inhibiting not only the action of the protease against synthetic fluorogenic substrates (Ki = 0.3 nM) but also blocking its processing of pro-interleukin-1beta (pro-IL-1ß). In addition, the peptide Ac-Asp-Glu-Val-Asp-glyoxal, which is based on the consensus cleavage sequence for caspase-3, is a potent inhibitor of this protease (Ki = 0.26 nM) yet only functions as a comparatively modest inhibitor of caspase-1 (Ki = 451 nM). Potent inhibitor sequences were also identified for caspases-6 and -8. However, the degree of discrimination between the family members is limited. The ability of Ac-Asp-Glu-Val-Asp-glyoxal to block caspase-3 like activity in whole cells and to delay the development of apoptosis was assessed. When tested against caspase-3 like activity in cell lysates, Ac-Asp-Glu-Val-Asp-glyoxal displayed effective inhibition similar to that observed against recombinant caspase-3. Treatment of whole cells with this potent caspase-3 inhibitor was however, not sufficient to significantly stall the development of apoptosis in-vitro.

Proteomics Identification of Azaspiracid Toxin Biomarkers in Blue Mussels, Mytilus edulis

Azaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Molecular & Cellular Proteomics 8: 1811-1822, 2009.

A rapid optical immunoassay for the screening of T-2 and HT-2 toxin in cereals and maize-based baby food

A rapid surface plasmon resonance (SPR) screening assay has been developed for the combined detection of T-2 and HT-2 toxins in naturally contaminated cereals using a sensor chip coated with an HT-2 toxin derivative and a monoclonal antibody. The antibody raised against HT-2 displayed high cross-reactivity with T-2 toxin while there was no cross-reaction observed with other commonly occurring trichothecenes. A simple extraction procedure using 40% methanol was applied to baby food, breakfast cereal, and wheat samples prior to biosensor analysis. Limits of detection (LOD) for each matrix were determined as 25 mu g kg(-1) for baby food and breakfast cereal and 26 mu g kg(-1) for wheat. Intra-assay precision (n = 6) was calculated for each matrix. The results were expressed as the relative standard deviation and determined as 2.8% (100 mu g kg(-1)) and 1.8% (200 mu g kg(-1)) in breakfast cereal, 4.6% (50 mu g kg(-1)) and 3.6% (100 mu g kg(-1)) in wheat and 0.97% (25 mu g kg(-1)) and 6.3% (50 mu g kg(-1)) in baby food. Between run precision (n = 3) performed at the same levels yielded relative standard deviations of 6.7% and 3.9% for breakfast cereals, 3.3% and 1.6% for wheat and 6.8% and 0.08% for baby food, respectively. (C) 2010 Elsevier B.V. All rights reserved.

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudonitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2-3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts. Published by Elsevier B.V.

Emerging cardiovascular actions of the incretin hormone glucagon-like peptide-1: potential therapeutic benefits beyond glycaemic control?

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by the small intestine in response to nutrient ingestion. It has wide-ranging effects on glucose metabolism, including stimulation of insulin release, inhibition of glucagon secretion, reduction of gastric emptying and augmentation of satiety. Importantly, the insulinotropic actions of GLP-1 are uniquely dependent on ambient glucose concentrations, and it is this particular characteristic which has led to its recent emergence as a treatment for type 2 diabetes. Although the major physiological function of GLP-1 appears to be in relation to glycaemic control, there is growing evidence to suggest that it may also play an important role in the cardiovascular system. GLP-1 receptors (GLP-1Rs) are expressed in the heart and vasculature of both rodents and humans, and recent studies have demonstrated that GLP-1R agonists have wide-ranging cardiovascular actions, such as modulation of heart rate, blood pressure, vascular tone and myocardial contractility. Importantly, it appears that these agents may also have beneficial effects in the setting of cardiovascular disease (CVD). For example, GLP-1 has been found to exert cardioprotective actions in experimental models of dilated cardiomyopathy, hypertensive heart failure and myocardial infarction (MI). Preliminary clinical studies also indicate that GLP-1 infusion may improve cardiac contractile function in chronic heart failure patients with and without diabetes, and in MI patients after successful angioplasty. This review will discuss the current understanding of GLP-1 biology, examine its emerging cardiovascular actions in both health and disease and explore the potential use of GLP-1 as a novel treatment for CVD.