AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog, Agalychnis callidryas

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Ranakinestatin-PPF from the Skin Secretion of the Fukien Gold-Striped Pond Frog, Pelophylax plancyi fukienensis: A Prototype of a Novel Class of Bradykinin B2 Receptor Antagonist Peptide from Ranid Frogs

The defensive skin secretions of many amphibians are a rich source of bradykinins and bradykinin-related peptides (BRPs). Members of this peptide group are also common components of reptile and arthropod venoms due to their multiple biological functions that include induction of pain, effects on many smooth muscle types, and lowering systemic blood pressure. While most BRPs are bradykinin receptor agonists, some have curiously been found to be exquisite antagonists, such as the maximakinin gene-related peptide, kinestatin—a specific bradykinin B₂-receptor antagonist from the skin of the giant fire-bellied toad, Bombina maxima. Here, we describe the identification, structural and functional characterization of a heptadecapeptide (DYTIRTRLHQGLSRKIV), named ranakinestatin-PPF, from the skin of the Chinese ranid frog, Pelophylax plancyi fukienensis, representing a prototype of a novel class of bradykinin B₂-receptor specific antagonist. Using a preconstricted preparation of rat tail arterial smooth muscle, a single dose of 10⁻⁶ M of the peptide effectively inhibited the dose-dependent relaxation effect of bradykinin between 10⁻¹¹ M and 10⁻⁵ M and subsequently, this effect was pharmacologically-characterized using specific bradykinin B₁- (desArg-HOE140) and B₂-receptor (HOE140) antagonists; the data from which demonstrated that the antagonism of the novel peptide was mediated through B₂-receptors. Ranakinestatin—PPF—thus represents a prototype of an amphibian skin peptide family that functions as a bradykinin B₂-receptor antagonist herein demonstrated using mammalian vascular smooth muscle.

Feleucins: Novel Bombinin Precursor-Encoded Nonapeptide Amides from the Skin Secretion of Bombina variegata

The first amphibian skin antimicrobial peptide (AMP) to be identified was named bombinin, reflecting its origin from the skin of the European yellow-bellied toad (Bombina variegata). Bombinins and their related peptides, the bombinin Hs, were subsequently reported from other bombinid toads. Molecular cloning of bombinin-encoding cDNAs from skin found that bombinins and bombinin Hs were coencoded on the same precursor proteins. Here, we report the molecular cloning of two novel cDNAs from a skin secretion-derived cDNA library of B. variegata whose open-reading frames each encode a novel bombinin (GIGGALLNVGKVALKGLAKGLAEHFANamide) and a C-terminally located single copy of a novel nonapeptide (FLGLLGGLLamide or FLGLIGSLLamide). These novel nonapeptides were named feleucin-BV1 and feleucin-BV2, respectively. The novel bombinin exhibited 89% identity to homologues from the toads, B. microdeladigitora and B. maxima. The feleucins exhibited no identity with any amphibian AMP archived in databases. Synthetic feleucins exhibited a weak activity against Staphylococcus aureus (128–256 mg/L) but feleucin-BV1 exhibited a synergistic action with the novel bombinin. The present report clearly demonstrates that the skin secretions of bombinid toads continue to represent a source of peptides of novel structure that could provide templates for the design of therapeutics.

Balteatide: A Novel Antimicrobial Decapeptide from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically-active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families, the dermatoxins, phylloxins, plasticins, distinctins and the medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus, the Gram-negative bacterium, Escherichia coli and the yeast, Candida albicans, and unusually for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

Feleucin-BO1: A Novel Antimicrobial Non-Apeptide Amide from the Skin Secretion of the Toad, Bombina orientalis, and Design of a Potent Broad-Spectrum Synthetic Analogue, Feleucin-K3

Feleucins-BV1 and -BV2 are recently-described prototypes of a novel antimicrobial nonapeptide (AMP) family identified in the skin secretion of the bombinid toad, Bombina variegata. They are encoded on different precursors that also encode a novel bombinin. Here we describe the identification of feleucin-BO1 (FLGLLGSLLamide) which is co-encoded with a different novel bombinin, named feleucin precursor-associated bombinin (FPA-bombinin-BO), from the skin secretion of Bombina orientalis. Synthetic feleucin-BO1 displayed activity against a reference Gram-positive bacterium. Staphylococcus aureus (MIC 34 μM) but was inactive (> 250 μM) against the Gram-negative bacterium, Escherichia coli, and the yeast, Candida albicans. This pattern of activity was similar to that of the prototypes. Design and synthesis of a cationicity-enhanced analogue, feleucin-K3 (F-K3), in which the amino acid residues at positions 3 (G), 6 (G) and 7 (S) of feleucin-BO1 were substituted with Lys (K) residues, resulted in a peptide with significantly-enhanced potency and spectrum of activity. The MICs of F-K3 against the reference microorganisms were 7 μM (S. aureus), 14 μM (E. coli) and 7 μM (C. albicans). These data indicate that the skin secretions of amphibians can continue to provide novel peptide templates for the rational design of analogues with possible therapeutic utility.

A Novel Bradykinin-Related Dodecapeptide (RVALPPGFTPLR) from the Skin Secretion of the Fujian Large-Headed Frog (Limnonectes fujianensis) Exhibiting Unusual Structural and Functional Features

Bradykinin-related peptides (BRPs) are significant components of the defensive skin secretions of many anuran amphibians, and these secretions represent the source of the most diverse spectrum of such peptides so far encountered in nature. Of the many families of bioactive peptides that have been identified from this source, the BRPs uniquely appear to represent homologues of counterparts that have specific distributions and receptor targets within discrete vertebrate taxa, ranging from fishes through mammals. Their broad spectra of actions, including pain and inflammation induction and smooth muscle effects, make these peptides ideal weapons in predator deterrence. Here, we describe a novel 12-mer BRP (RVALPPGFTPLR-RVAL-(L1, T6, L8)-bradykinin) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis). The C-terminal 9 residues of this BRP (-LPPGFTPLR) exhibit three amino acid substitutions (L/R at Position 1, T/S at Position 6 and L/F at Position 8) when compared to canonical mammalian bradykinin (BK), but are identical to the kinin sequence present within the cloned kininogen-2 from the Chinese soft-shelled turtle (Pelodiscus sinensis) and differ from that encoded by kininogen-2 of the Tibetan ground tit (Pseudopodoces humilis) at just a single site (F/L at Position 8). These data would imply that the novel BRP is an amphibian defensive agent against predation by sympatric turtles and also that the primary structure of the avian BK, ornithokinin (RPPGFTPLR), is not invariant within this taxon. Synthetic RVAL-(L1, T6, L8)-bradykinin was found to be an antagonist of BK-induced rat tail artery smooth muscle relaxation acting via the B2-receptor.

A Review on Bradykinin-Related Peptides Isolated from Amphibian Skin Secretion

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure–function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future.

Whey proteins have beneficial effects on intestinal enteroendocrine cells stimulating cell growth and increasing the production and secretion of incretin hormones

Whey protein has been indicated to curb diet-induced obesity, glucose intolerance and delay the onset of type 2 diabetes mellitus. Here the effects of intact crude whey, intact individual whey proteins and beta-lactoglobulin hydrolysates on an enteroendocrine (EE) cell model were examined. STC-1 pGIP/neo cells were incubated with several concentrations of yogurt whey (YW), cheese whey (CW), beta-lactoglobulin (BLG), alpha-lactalbumin (ALA) and bovine serum albumin (BSA). The findings demonstrate that BLG stimulates EE cell proliferation, and also GLP-1 secretion (an effect which is lost following hydrolysis with chymotrypsin or trypsin). ALA is a highly potent GLP-1 secretagogue which also increases the intracellular levels of GLP-1. Conversely, whey proteins and hydrolysates had little impact on GIP secretion. This appears to be the first investigation of the effects of the three major proteins of YW and CW on EE cells. The anti-diabetic potential of whey proteins should be further investigated.

Development of a doped titania immobilised thin film multi tubular photoreactor

This paper describes a novel doped titania immobilised thin film multi tubular photoreactor which has been developed for use with liquid, vapour or gas phase media. In designing photocatalytic reactors measuring active surface area of photocatalyst within the unit is one of the critical design parameters. This dictate greatly limits the applicability of any semi-conductor photocatalyst in industrial applications, as a large surface area equates to a powder catalyst. This demonstration of a thin film coating, doped with a rare earth element, novel photoreactor design produces a photocatalytic degradation of a model pollutant (methyl orange) which displayed a comparable degradation achieved with P25 TiO2. The use of lanthanide doping is reported here in the titania sol gel as it is thought to increase the electron hole separation therefore widening the potential useful wavelengths within the electromagnetic spectrum. Increasing doping from 0.5% to 1.0% increased photocatalytic degradation by ∼17% under visible irradiation. A linear relationship has been seen between increasing reactor volume and degradation which would not normally be observed in a typical suspended reactor system. © 2012 Elsevier B.V.