The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

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

Antimicrobial activity of truncated alpha-defensin (human neutrophil peptide (HNP)-1) analogues without disulphide bridges.

Antimicrobial peptides play an important role in host defence, particularly in the oral cavity where there is constant challenge by microorganisms. The a-defensin antimicrobial peptides comprise 30–50% of the total protein in the azurophilic granules of human neutrophils, the most abundant of which is human neutrophil peptide 1 (HNP-1). Despite its antimicrobial activity, a limiting factor in the potential therapeutic use of HNP-1 is its chemical synthesis with the correct disulphide topology. In the present study, we synthesised a range of truncated defensin analogues lacking disulphide bridges. All the analogues were modelled on the C-terminal region of HNP-1 and their antimicrobial activity was tested against a range of microorganisms, including oral pathogens. Although there was variability in the antimicrobial activity of the truncated analogues synthesised, a truncated peptide named 2Abz23S29 displayed a broad spectrum of antibacterial activity, effectively killing all the bacterial strains tested. The finding that truncated peptides, modelled on the C-terminal ß-hairpin region of HNP-1 but lacking disulphide bridges, display antimicrobial activity could aid their potential use in therapeutic interventions.

Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning.

The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.

Role of creatine supplementation on exercise-induced cardiovascular function and oxidative stress

Many degenerative diseases are associated with increased oxidative stress. Creatine has the potential to act as an indirect and direct antioxidant; however, limited data exist to evaluate the antioxidant capabdities of creatine supplementation within in vivo human systems. This study aimed to investigate the effects of oral creatine supplementation on markers of oxidative stress and antioxidant defenses following exhaustive cycling exercise. Following preliminary testing and two additional familiarization sessions, 18 active males repeated two exhaustive incremental cycling trials (T1 and T2) separated by exactly 7 days. The subjects were assigned, in a double-blind manner, to receive either 20 g of creatine (Cr) or a placebo (P) for the 5 days preceding T2. Breath-by-breath respiratory data and heart rate were continually recorded throughout the exercise protocol and blood samples were obtained at rest (preexercise), at the end of exercise (postexercise), and the day following exercise (post24 h). Serum hypdroperoxide concentrations were elevated at postexercise by 17 +/- 5% above preexercise values (p = 0.030). However, supplementation did not influence lipid peroxidation (serum hypdroperoxide concentrations), resistance of low density lipoprotein to oxidative stress (t(1/2max) LDL oxidation) and plasma concentrations of non-enzymatic antioxidants (retinol, alpha-carotene, beta-carotene, alpha-tocopherol, gamma-tocopherol, lycopene and vitamin Q. Heart rate and oxygen uptake responses to exercise were not affected by supplementation. These findings suggest that short-term creatine supplementation does not enhance non-enzymatic antioxidant defence or protect against lipid peroxidation induced by exhaustive cycling in healthy males.

Carrying religion into a secularising Europe: Montserratian migrants' experiences of global processes in British methodism

Migrants to Europe often perceive themselves as entering a secular society that threatens their religious identities and practices. Whilst some sociological models present their responses in terms of cultural defence, ethnographic analysis reveals a more complex picture of interaction with local contexts. This essay draws upon ethnographic research to explore a relatively neglected situation in migration studies, namely the interactions between distinct migration cohorts - in this case, from the Caribbean island of Montserrat, as examined through their experiences in London Methodist churches. It employs the ideas of Weber and Bourdieu to view these migrants as 'religious carriers', as collective and individual embodiments of religious dispositions and of those socio-cultural processes through which their religion is reproduced. Whilst the strategies of the cohort migrating after the Second World War were restricted through their marginalised social status and experience of racism, the recent cohort of evacuees fleeing volcanic eruptions has had greater scope for strategies which combat secularisation and fading Methodist identity.

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.

Antimicrobial peptides from the skin secretion of the Wuyi Mountain torrent frog, Amolops wuyiensis: An inhabitant of a pristine aquatic environment

The antimicrobial peptides of amphibian skin secretions are proposed to aid survival in microbe-rich environments. While many amphibians inhabit such environments, other such as the Wuyi Mountain torrent frog, Amolops wuyiensis, live in pristine waters flowing from underground mountain springs. This species thus represents an interesting model in which to study antimicrobial peptides. “Shotgun” cloning of a skin-derived cDNA library from this species identified transcripts encoding a brevinin-1 and a ranatuerin-2. Peptides with coincident molecular masses to both predicted mature peptides were identified in HPLC fractions of skin secretion. Synthetic replicates of both peptides were generated by solid-phase peptide synthesis and tested for activity using Staphylococcus aureus, Escherichia coli and Candida albicans. The brevinin was found to be broad-spectrum and potent with minimum inhibitory concentrations (MICs) of 24 µM (Sa), 5 µM (Ec) and 20 µM (Ca). In contrast, the ranatuerin was less effective and of narrower spectrum with an MIC > 200 µM for Sa, 40 µM (Ec) and 120 µM (Ca). Thus this species of amphibian that lives in a pristine environment does indeed possess at least one potent and broad-spectrum antimicrobial peptide in its skin secretion arsenal. This phenomenon could be explained in several ways. Firstly, it may represent an ancestral peptide required when the stem species inhabited microbe-rich environments. However, there is mounting evidence for the second reason, that suggests the function of such peptides is not primarily in antimicrobial defence.

Costs of resistance: genetic correlations and potential trade-offs in an insect immune system

Theory predicts that natural selection will erode additive genetic variation in fitness-related traits. However, numerous studies have found considerable heritable variation in traits related to immune function, which should be closely linked to fitness. This could be due to trade-offs maintaining variation in these traits. We used the Egyptian cotton leafworm, Spodoptera littoralis, as a model system to examine the quantitative genetics of insect immune function. We estimated the heritabilities of several different measures of innate immunity and the genetic correlations between these immune traits and a number of life history traits. Our results provide the first evidence for a potential genetic trade-off within the insect immune system, with antibacterial activity (lysozyme-like) exhibiting a significant negative genetic correlation with haemocyte density, which itself is positively genetically correlated with both haemolymph phenoloxidase activity and cuticular melanization. We speculate on a potential trade-off between defence against parasites and predators, mediated by larval colour, and its role in maintaining genetic variation in traits under natural selection.

The use of process simulation methods in support of organisational learning in availability contracting

Traditional business models in the aerospace industry are based on a conventional supplier to customer relationship based on the design, manufacture and subsequent delivery of the physical product. Service provision, from the manufacturer's perspective, is typically limited to the supply of procedural documentation and the provision of spare parts to the end user as the product passes through the latter stages of its intended lifecycle. Challenging economic and political conditions have resulted in end users re-structuring their core business activities, particularly in the defence sector. This has resulted in the need for original equipment manufacturers (OEMs) to integrate and manage support service activities in partnership with the customer to deliver platform availability. This improves the probability of commercial sustainability for the OEM through shared operational risks while reducing the cost of platform ownership for the customer. The need for OEMs to evolve their design, manufacture and supply strategies by focusing on customer requirements has revealed a need for reconstruction of traditional internal behaviours and design methodologies. Application of organisational learning is now a well recognised principle for innovative companies to achieve long term growth and sustained technical development, and hence, greater market command. It focuses on the process by which the organisation's knowledge and value base changes, leading to improved problem solving ability and capacity for action. From the perspective of availability contracting, knowledge and the processes by which it is generated, used and retained, become primary assets within the learning organisation. This paper will introduce the application of digital methods to asset management by demonstrating how the process of learning can benefit from a digital approach, how product and process design can be integrated within a virtual framework and finally how the approach can be applied in a service context.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Suppressors of cytokine signalling (SOCS): putative modulators of cytokine bioactivity in health and disease

Cytokines are important modulators of homeostatic processes such as development, haematopoiesis and host defence, A recently identified family of proteins, the supressors of cytokine signalling (SOCS) act as negative regulators of the key cytokine-activated signalling pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade, In the current review, the discovery, structural features, regulation of expression, mechanisms of JAK/STAT inhibition and putative role in health and disease of the SOCS family are discussed.

Antimicrobial peptides and alytesin are co-secreted from the venom of the Midwife toad, Alytes maurus (Alytidae, Anura): Implications for the evolution of frog skin defensive secretions

The skin secretions of frogs and toads (Anura) have long been a known source of a vast abundance of bioactive substances. In the past decade, transcriptome data of the granular glands of anuran skin has given new impetus to investigations of the putative constituent peptides. Alytes obstetricans was recently investigated and novel peptides with antimicrobial activity were isolated and functionally characterised. However, genetic data for the evolutionarily ancient lineage to which Alytes belongs (midwife toads; Alytidae) remains unavailable.

Here we present the first such genetic data for Alytidae, derived via the granular gland transcriptome of a closely-related species of midwife toad, Alytes maurus. First, we present nucleotide sequences of the entire peptide precursors for four novel antimicrobial peptides (AMPs). The two precursors resemble those from Bombinatoridae in both their structural architecture and amino acid sequence. Each precursor comprises two AMPs as tandem repeats, with a member of the alyteserin-1 family (alyteserin-1Ma: GFKEVLKADLGSLVKGIAAHVAN-NH2 or alyteserin-1Mb: GFKEVLKAGLGSLVKGIPAHVAN-NH2) followed by its corresponding member from the alyteserin-2 family (alyteserin-2Ma: FIGKLISAASGLLSHL-NH2 or alyteserin-2Mb: ILGAIIPLVSGLLSHL-NH2). Synthetic replicates of the four AMPs possessed minimal inhibitory concentrations (MICs) ranging from 9.5 to 300 µM, with the most potent being alyteserin-2Ma. Second, we also cloned the cDNA encoding an alytesin precursor, with the active alytesin exhibiting high sequence identity to bombesin-related peptides from other frogs. All putative mature peptide sequences were confirmed to be present in the skin secretion via LC/MS.

The close structural resemblance of the alyteserin genes that we isolated for A. maurus with those of Bombina provide independent molecular evidence for a close evolutionary relationship between these genera as well as more support for the convergent evolution of the AMP system within anurans. In contrast to the more evolutionarily conserved nature of neuropeptides (including alytesin, which we also isolated), the more variable nature of the AMP system together with the sporadic distribution of AMPs among anuran amphibians fuels in part our hypothesis that the latter system was co-opted secondarily to fulfil a function in the innate immune system, having originally evolved for defence against potential macropredators.