Kassina senegalensis skin tachykinins: Molecular cloning of kassinin and (Thr2, Ile9)-kassinin biosynthetic precursor cDNAs and comparative bioactivity of mature tachykinins on the smooth muscle of rat urinary bladder

Tachykinins are among the most widely-studied families of regulatory peptides characterized by a highly-conserved C-terminal -Phe-X-Gly-Leu-Met.amide motif, which also constitutes the essential bioactive core. The amphibian skin has proved to be a rich source of these peptides with physalaemin from the skin of Physalaemus fuscomaculatus representing the archetypal aromatic tachykinin (X = Tyr or Phe) and kassinin from the skin of Kassina senegalensis representing the archetypal aliphatic tachykinin in which X = Val or Ile. Despite the primary structures of both mature peptides having been known for at least 30 years, neither the structures nor organizations of their biosynthetic precursors have been reported. Here we report the structure and organization of the biosynthetic precursor of kassinin deduced from cDNA cloned from a skin secretion library. In addition, a second precursor cDNA encoding the novel kassinin analog (Thr2, Ile9)-kassinin was identified as was the predicted mature peptide in skin secretion. Both transcripts exhibited a high degree of nucleotide sequence similarity and of open-reading frame translated amino acid sequences of putative precursor proteins. The translated preprotachykinins each consisted of 80 amino acid residues encoding single copies of either kassinin or its site-substituted analog. Synthetic replicates of each kassinin were found to be active on rat urinary bladder smooth muscle at nanomolar concentrations. The structural organization of both preprotachykinins differs from that previously reported for those of Odorrana grahami skin indicating a spectrum of diversity akin to that established for amphibian skin preprobradykinins.

Langerhans cell (LC) proliferation mediates neonatal development, homeostasis, and inflammation-associated expansion of the epidermal LC network.

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.

Discovery of multiple neuropeptide families in the phylum Platyhelminthes

Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Nigrocin-2 peptides from Chinese Odorrana frogs – integration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis

Peptidomics is a powerful set of tools for the identification, structural elucidation and discovery of novel regulatory peptides and for monitoring the degradation pathways of structurally and catalytically important proteins. Amphibian skin secretions, arising from specialized granular glands, often contain complex peptidomes containing many components of entirely novel structure and unique site-substituted analogues of known peptide families. Following the discovery that the granular gland transcriptome is present in such secretions in a PCR-amenable form, we designed a strategy for peptide structural characterization involving the integration of ‘shotgun’ cloning of cDNAs encoding peptide precursors, deduction of putative bioactive peptide structures, and confirmation of these structures using tandem MS/MS sequencing. Here, we illustrate this strategy by means of elucidation of the primary structures of nigrocin-2 homologues from the defensive skin secretions of four species of Chinese Odorrana frogs, O. schmackeri, O. livida, O. hejiangensis and O. versabilis. Synthetic replicates of the peptides were found to possess antimicrobial activity. Nigrocin-2 peptides occur widely in the skin secretions of Asian ranid frogs and in those of the Odorrana group, and are particularly well-represented and of diverse structure in some species. Integration of the molecular analytical technologies described provides a means for rapid structural characterization of novel peptides from complex natural libraries in the absence of systematic online database information.

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

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

Molecular cloning and deduced organization of the pHpG/CNP precursor from the venom of the African forest viper, Atheris squamigera

The discovery that the hypotensive sequela of envenomation by the South American viper, Bothrops jararaca, was mediated by peptides, represented a milestone in drug discovery research that led to the introduction of ACE inhibitors. These bradykinin-potentiating peptides (BPPs) have been found in the venoms of many species of viper and molecular cloning of biosynthetic precursors has revealed that each encodes several different BPPs in tandem with a single copy of a C-type natriuretic peptide (CNP) located at the C-terminus. Venoms of the African forest vipers (Atheris) have been poorly studied possibly because they do not represent a major danger to humans. However, initial studies have indicated that they contain some of the “classical” protein toxins of viper venoms and a novel class of peptide, the polyglycine/polyhistidine (pGpH) peptides. These peptides occur in several molecular forms with different numbers of repetitive glycine and histidine repeats. We have cloned the biosynthetic precursor of A. squamigera pGpH peptides from a venom-derived cDNA library and have confirmed that a single copy of CNP is located at the C-terminus and additionally that, like BPPs in other vipers, pGpH peptides are encoded in tandem within this single precursor. Solid phase peptide synthesis of pGpH peptides has proven to be extremely difficult but is progressing and acquisition of synthetic replicates of each peptide is a necessary prerequisite for systematic pharmacological characterisation as establishment of a biological function for these peptides remains elusive. pGpH peptides may prove to play a role as fundamental as that of the BPPs.

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.

Atmospheric parameters and rotational velocities for a sample of Galactic B-type supergiants

High-resolution optical spectra of 57 Galactic B-type supergiant stars have been analysed to determine their rotational and macroturbulent velocities. In addition, their atmospheric parameters (effective temperature, surface gravity and microturbulent velocity) and surface nitrogen abundances have been estimated using a non-local thermodynamic equilibrium grid of model atmospheres. Comparisons of the projected rotational velocities have been made with the predictions of stellar evolutionary models and in general good agreement was found. However, for a small number of targets, their observed rotational velocities were significantly larger than predicted, although their nitrogen abundances were consistent with the rest of the sample. We conclude that binarity may have played a role in generating their large rotational velocities. No correlation was found between nitrogen abundances and the current projected rotational velocities. However, a correlation was found with the inferred projected rotational velocities of the main-sequence precursors of our supergiant sample. This correlation is again in agreement with the predictions of single star evolutionary models that incorporate rotational mixing. The origin of the macroturbulence and microturbulent velocity fields is discussed and our results support previous theoretical studies that link the former to subphotospheric convection and the latter to non-radial gravity mode oscillations. In addition, we have attempted to identify differential rotation in our most rapidly rotating targets.

Synthesis and Reactions of Enantiopure Substituted Benzene cis-Hexahydro-1,2-diols

Enantiopure cis-dihydro-1,2-diol metabolites, obtained from toluene dioxygenase-catalysed cis-dihydroxylation of six monosubstituted benzene substrates, have been converted to their corresponding cis-hexahydro-12-diol derivatives by catalytic hydrogenation via their cis-tetrahydro-1,2-diol intermediates. Optimal reaction conditions for total catalytic hydrogenation of the cis-dihydro-1,2-diols have been established using six heterogeneous catalysts. The relative and absolute configurations of the resulting benzene cis-hexahydro-1,2-diol products have been unequivocally established by X-ray crystallography and NMR spectroscopy. Methods have been developed to obtain enantiopure cis-hexahydro-1,2-diol diastereoisomers, to desymmetrise a meso-cis-hexahydro-1,2-diol and to synthesise 2-substituted cyclohexanols. The potential of these enantiopure cyclohexanols as chiral reagents was briefly evaluated through their application in the synthesis of two enantiomerically enriched phosphine oxides from the corresponding racemic phosphine precursors.

A Population-Based Study of IGF Axis Polymorphisms and the Esophageal Inflammation, Metaplasia, Adenocarcinoma Sequence

BACKGROUND & AIMS: Insulin-like growth factor (IGF) axis plays a key role in cell development, proliferation, and survival and is implicated in the etiology of several cancers. Few studies have examined the relationship between genetic variation of this axis and esophageal adenocarcinoma (EAC) or its precursors. METHODS: In a population-based case-control study, we investigated the association of common polymorphisms of IGF-1, IGF-2, IGF-1 receptor, IGF binding protein -3, growth hormones (GH) 1 and GH2, and GH receptor with reflux esophagitis (RE), Barrett esophagus (BE), and EAC. Two hundred and thirty RE, 224 BE, 227 EAC cases, and 260 controls were studied. Gene polymorphisms were identified using publicly available online resources; 102 IGF axis tag and putatively functional single-nucleotide polymorphisms (SNPs) were analyzed using MassARRAY iPLEX and Taqman assays. Results were analyzed using Haploview.
RESULTS: Three polymorphisms were disease-associated. IGF1 SNP rs6214 was associated with BE (adjusted P = .039). Using GG genotype as reference, odds ratio for BE in AA (wild-type) was 0.43 (95% confidence interval [CI], 0.24-0.75). GH receptor SNP rs6898743 was associated with EAC (adjusted P = .0112). With GG as reference, odds ratio for EAC in CC (wildtype) genotype was 0.42 (95% CI, 0.23-0.76). IGF1 (CA)(17) 185-bp allele was associated with RE (adjusted P = .0116). Using IGF1(non17) as reference, odds ratio for RE in IGF1(17) carriers was 7.29 (95% CI, 1.57-46.7).
CONCLUSIONS: In this study, 3 polymorphisms of IGF genes were associated with EAC or its precursors. These polymorphisms may be markers of disease risk; independent validation of our findings is required. These results suggest the IGF pathway is involved in EAC development.

Negotiating Civic Space in Belfast or The Tricolour: Here Today, Gone Tomorrow’

What role does public civic space in Belfast city centre play in the negotiation of different political identities within the city? Focusing on key public events in this space the paper traces shifts in identity practices and focuses on negotiations over the uses of public space associated with Irish nationalism and British unionism. This, it is argued, gives a more sophisticated understanding of different types of ‘shared space’. The events probed are seen as precursors and possibly drivers of political change. It is concluded that the increased sharing of civic space has probably contributed to improved political relations within the city, though there remains the challenge of
understanding how public space might more effectively be used to influence
relationships between the city’s political identities in the longer term.

The effect of simvastatin on bone formation and ceramic resorption in a peri-implant defect model

Experimental use of statins as stimulators of bone formation suggests they may have widespread applicability in the field of orthopaedics. With their combined effects on osteoblasts and osteoclasts, statins have the potential to enhance resorption of synthetic materials and improve bone ingrowth. In this study, the effect of oral and local administration of simvastatin to a 0 tricalcium phosphate (beta TCP)-filled defect around an implant was compared with recombinant human bone morphogenetic protein 2 (rhBMP2). On hundred and sixty-two Sprague-Dawley rats were assigned to treatment groups: local application of 0.1, 0.9 or 1.7 mg of simvastatin, oral simvastatin at 5, 10 or 50 mg kg(-1) day(-1) for 20 days, local delivery of I or 10 mu g of rhBMP2, or control. At 6 weeks rhBMP2 increased serum tartrate-resistant acid phosphatase 5b levels and reduced PTCP area fraction, particle size and number compared with control, suggesting increased osteoclast activity. There was reduced stiffness and increased mechanical strength with this treatment. Local simvastatin resulted in a decreased mineral apposition rate at 6 weeks and increased fibrous area fraction, PTCP area fraction, particle size and number at 26 weeks. Oral simvastatin had no effect compared with control. Local application of rhBMP2 increased resorption and improved mechanical strength whereas simvastatin was detrimental to healing. Oral simvastatin was ineffective at promoting either ceramic resorption or bone formation. The effect of statins on the repair of bone defects with graft substitute materials is influenced by its bioavailability. Thus, further studies on the optimal delivery system are needed. (C) 2007 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The effect of osteogenic growth factors on bone growth into a ceramic filled defect around an implant

Currently available synthetic bone substitutes perform poorly compared to autograft. It is hoped that by adding osteogenic growth factors to the materials, new bone formation could be increased and the clinical outcome improved. In this study, IGF-1, bFGF and TGFbeta1, alone and in combination, were absorbed onto a carrier of P-tricalcium phosphate (PTCP) and implanted into a defect around a hydroxyapatite-coated, stainless steel implant in the proximal tibia of rat in a model of revision arthroplasty. Animals were sacrificed at 6 and 26 weeks for routine histology and histomorphometry and mechanical push out tests. The results show that only bFGF had a significant effect on ceramic resorption. The groups that received bFGF and bFGF in combination with TGFbeta1 had smaller and fewer betaTCP particles remaining in the defect at 6 and 26 weeks. No growth factor combination significantly enhanced new bone formation or the mechanical strength of the implant. These results indicate that, of the growth factors tested, only bFGF had any beneficial effect on the host response to the implant, perhaps by delaying osteoblast differentiation and thereby prolonging osteoclast access to the ceramic. (C) 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.