Vasorelaxin: A Novel Arterial Smooth Muscle-Relaxing Eicosapeptide from the Skin Secretion of the Chinese Piebald Odorous Frog (Odorrana schmackeri)

The defensive skin secretions of amphibians are a rich resource for the discovery of novel, bioactive peptides. Here we report the identification of a novel vascular smooth muscle-relaxing peptide, named vasorelaxin, from the skin secretion of the Chinese piebald odorous frog, Odorrana schmackeri. Vasorelaxin consists of 20 amino acid residues, SRVVKCSGFRPGSPDSREFC, with a disulfide-bridge between Cys-6 and Cys-20. The structure of its biosynthetic precursor was deduced from cloned skin cDNA and consists of 67 amino acid residues encoding a single copy of vasorelaxin (vasorelaxin, accession number: HE860494). Synthetic vasorelaxin caused a profound relaxation of rat arterial smooth muscle with an EC50 of 6.76 nM.

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.

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.

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri) skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP) and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues) or CGRPs (37 amino acids) and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilized skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides 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, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha(1)-adrenergic receptor activation: Evidence for a salt bridge as the initiating process

alpha(1)-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (Porter et al., 1996). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha(1)-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha(1b)-AR and in Rat-1 fibroblasts stably transfected with the human alpha(1a)-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha(1)-AR agonists and this effect was fully inhibited by the alpha(1)-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha(1)-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha(1)-ARs with a K-i of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha(1)-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha(1)-AR activation.

Synergistic effects on gene delivery - co-formulation of small disulfide-linked dendritic polycations with Lipofectamine 2000 (TM)

This paper describes the application of gene delivery vectors based on connecting together two well-defined low-generation poly(L-lysine) (PLL) dendrons using a disulfide-containing linker unit. We report that the transfection ability of these vectors in their own right is relatively low, because the low-generation number limits the endosomal buffering capacity. Importantly, however, we demonstrate that when applied in combination with Lipofectamine 2000 (TM), a vector from the cationic lipid family, these small cationic additives significantly enhance the levels of gene delivery (up to four-fold). Notably, the cationic additives have no effect on the levels of transfection observed with a cationic polymer, such as DEAE dextran. We therefore argue that the synergistic effects observed with Lipofectamine 2000 (TM) arise as a result of combining the delivery advantages of two different classes of vector within a single formulation, with our dendritic additives providing a degree of pH buffering within the endosome. As such, the data we present indicate that small dendritic structures, although previously largely overlooked for gene delivery owing to their inability to transfect in their own right, may actually be useful well-defined additives to well-established vector systems in order to enhance the gene delivery payload.

The hydraulics and resulting bed scour within the vicinity of Submerged Single Span Arch Bridges

As a consequence of climate change there is now a more frequent occurrence of extreme rainfall events where, with higher rates of urbanisation, the built environment has become increasingly affected by flooding.. This is of particular importance in relation to the stability of bridge structures that span rivers and canals etc. In November 2009, the UK and Ireland were subjected to extraordinarily severe weather conditions for several days. The rainfall was logged as the highest level of rainfall ever recorded within the UK, and as a direct consequence, unprecedented flooding occurred in Cumbria. This flooding led to the collapse of three road bridges which were generally 19th century masonry arch bridges, with relatively shallow foundations. In the UK, knowledge of the combined effect of bridge scouring and inundation has been not been particularly widely studied. Research carried out by Hamill et al [1] considered the hydraulic analysis of single arch bridges under flood conditions, but no consideration was given towards the likely damage to these structures due to scouring. Prior to this, Bierry and Delleur [2] produced a classic paper in predicting the discharge downstream of an inundated arch, focussing on predicting afflux as opposed to bridge scour. Further work on backwater effects was carried out by Martin-Vide & Prio [3] in semi-circular arch bridges. Both pressurized and free-surface flows at the bridge were investigated. Flows on a mobile bed in clear-water conditions were compared to those with a rigid bed, but no predictive equation for scour under pressurised conditions was considered. This paper will present initial findings from an experimental investigation into the effects of surcharged flow and subsequent scour within the vicinity of single span arch bridges. Velocities profiles will be shown within the vicinity of the arch, in addition to the depth of clear water scour, for a series of flows and model spans. The data will be presented, where results will be correlated to the most recent predictive equations that are proposed.

Next generation Bridge weigh-in-motion using fibre optic sensors

There have been over 3000 bridge weigh-in-motion (B-WIM) installations in 25 countries worldwide, this has led vast improvements in post processing of B-WIM systems since its introduction in the 1970’s. Existing systems are based on electrical resistance strain gauges which can be prohibitive in achieving data for long term monitoring of rural bridges due to power consumption. This paper introduces a new low-power B-WIM system using fibre optic sensors (FOS). The system consisted of a series of FOS which were attached to the soffit of an existing integral bridge with a single span of 19m. The site selection criteria and full installation process has been detailed in the paper. A method of calibration was adopted using live traffic at the bridge site and based on this calibration the accuracy of the system was determined. New methods of axle detection for B-WIM were investigated and verified in the field.

Bridge weigh-in-motion system and Structural Health Monitoring using fiber optic sensors

There have been over 3000 bridge weigh-in-motion (B-WIM) installations in 25 countries worldwide, this has led vast improvements in post processing of B-WIM systems since its introduction in the 1970’s. This paper introduces a new low-power B-WIM system using fibre optic sensors (FOS). The system consisted of a series of FOS which were attached to the soffit of an existing integral bridge with a single span of 19m. The site selection criteria and full installation process has been detailed in the paper. A method of calibration was adopted using live traffic at the bridge site and based on this calibration the accuracy of the system was determined.