D. David Smith

Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda

A peptide related to urotensin II has been isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the peptide was established as Ala-Gly-Asn-Leu-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val and this sequence was confirmed by chemical synthesis. Frog urotensin II contains an additional amino acid residue compared with fish urotensin II peptides but the structure of the cyclic region of the molecule has been fully conserved. The data show that urotensin II is not confined to the caudal neurosecretory system of fish but is present in the central nervous system of a tetrapod.

Ranakinin: A novel NK1 tachykinin receptor agonist isolated with neurokinin B from the brain of the frog Rana ridibunda

An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P‐like immunoreactivity, measured with an antiserum directed against the COOH‐terminal region of mammalian substance p and neurokinin b‐like immurtoreactivity, measured with an antiserum directed against the NH2‐terminus of neurokinin B. The primary structure of the substance p‐related peptide (ranakinin) was established as: Lys‐Pro‐Asn‐Pro‐Glu‐Arg‐Phe‐Tyr‐Gly‐Leu‐Met‐NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin b‐related peptide was established as: Asp‐Met‐His‐Asp‐Phe‐Phe‐Val‐Gly‐Leu‐Met‐NH2. This amino acid sequence is the same as that of mammalian neurokinin B.

Purification and Characterization of Urotensin II and Parvalbumin from an Elasmobranch Fish, Scyliorhinus canicula (Common Dogfish)

The caudal portion of the spinal cord of elasmobranch fish incorporates a diffuse neuroendocrine system. Using an antiserum raised against urotensin II from a teleost fish (goby) to facilitate purification, a peptide structurally related to urotensin II was isolated in pure form from an extract of neuroendocrine tissue from the spinal cord of the European common dogfish, Scyliorhinus canicula. The primary structure of the peptide was established as: Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. The amino acid sequence was confirmed by chemical synthesis. A comparison of this sequence with those of the known teleost urotensin II peptides shows that the cyclic region of the molecule has been fully conserved between species and suggests that the presence of an acidic residue at position 5 and a hydrophobic residue at position 12 are important features for the biological activity of the peptide. The dogfish spinal cord extract also contained a high concentration of the calcium-binding protein, parvalbumin and the amino acid sequence at its NH2 terminus [residues (1-50)] was determined.

Membrane Protease Degradomics: Proteomic Identification and Quantification of Cell Surface Protease Substrates

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

Pharmacological Characterization of Novel α-Calcitonin Gene-Related Peptide (CGRP) Receptor Peptide Antagonists That Are Selective for Human CGRP Receptors

Human α-calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that produces a variety of cardiovascular and other effects via activation of specific CGRP receptors that produce cAMP. Functional CGRP receptors are a heterodimeric complex composed of the heptahelical calcitonin receptor-like receptor and the single transmembrane receptor activity-modifying protein 1. Based on the known structures of the antagonist CGRP(8–37) and the human CGRP receptor, we designed novel CGRP receptor peptide antagonists with modifications to promote high affinity and selectivity for human CGRP receptors. Antagonist affinity (KB) at CGRP receptors was determined using the mouse thoracic aorta and human SK-N-MC cells. In aorta, CGRP(8–37), [N-α-benzoyl]human α-CGRP(8–37) [bzl-CGRP(8–37)], and [N-α-benzoyl-His10-benzyl]human α-CGRP(8–37) [bzl-bn-CGRP(8–37)] caused rightward shifts in the concentration-response relaxation curve for CGRP with KB values of 1000, 88, and 50 nM, respectively. In human SK-N-MC cells, CGRP(8–37), bzl-CGRP(8–37), and bzl-bn-CGRP(8–37) caused rightward shifts in the concentration-response curve for CGRP-stimulated cAMP production with KB values of 797, 15, and 0.63 nM, respectively. Thus, CGRP(8–37) had the same affinity for human and mouse CGRP receptors, whereas bzl-CGRP(8–37) and bzl-bn-CGRP(8–37) displayed 6- and 80-fold higher affinities, respectively, for human CGRP receptors. In addition, the selectivity of the antagonists for human CGRP receptors was highly correlated with the antagonist hydrophobicity index. These relatively high-affinity, species-selective peptide antagonists provide novel tools to differentiate structural and functional features that are unique to the human CGRP receptor. Thus, these analogs may be useful compounds for development of drugs to treat migraine headache and other cardiovascular diseases.

Measurement of T-kinin in rat plasma using a specific radioimmunoassay

T-kinin (Ile-Ser-Bradykinin) has been isolated only from the plasma of the rat and it is unclear whether the peptide, or its biosynthetic precursor, T-kininogen, circulates in the human. An NH2-terminally directed antiserum to T-kinin was raised in rabbits using an immunogen prepared by coupling the free -SH group of T-kinin extended from its COOH-terminus by a cysteinyl residue to an -NH2 group on human serum albumin. A radioimmunoassay was developed using this antiserum and 125I-labelled [Tyr10]T-kinin as tracer that was sensitive (least-detectable concentration 3 fmol/tube) and relatively specific for T-kinin (cross-reactivity with bradykinin and kallidin less than 1%). Treatment of rat plasma with an excess of trypsin in the presence of a kininase inhibitor generated T-kinin immunoreactivity equivalent to 455 +/- 71 pmol/ml (mean +/- S.E.M.; n = 9) and this immunoreactivity was eluted from a reversed-phase HPLC column as a single peak with the same retention time as synthetic T-kinin. In contrast, treatment of plasma from healthy human subjects (n = 8) and from patients (n = 8) with inflammation due to acute or chronic gastrointestinal disease under the same conditions did not generate any detectable T-kinin immunoreactivity. It is concluded, therefore, that T-kininogen, the biosynthetic precursor of T-kinin in the rat, is either absent from the plasma of human subjects or is present in a concentration less than 30 fmol/ml. Similarly, T-kininogen is probably not an acute phase reactant in humans.

Effect of solvent on racemization in carbodiimide mediated solid phase fragment condensations

Abstract The Izumiya tripeptide was used to assess racemization in solid-phase fragment condensations. Boc-Gly-Ala-OH 1 was coupled to Leu-PAM resin DIC in a variety of solvents, both with and without HOBt. After cleavage from the resin, the extent of racemization was determined using C18 RP-HLPC to separate the epimers. Solvents used were DMF, NMP, TFE and each of these as mixtures with DCM. Also tested was a mixture of NMP and DMSO (85:15). Couplings in DMF/DCM (1:1) and NMP/DCM (1:1) in the presence of HOBt were in excess of 98% and racemization was undetectable. (

A tale of two soles: sociomechanical and biomechanical considerations in diabetic limb salvage and amputation decision-making in the worst of times

Foot ulcerations complicated by infection are the major cause of limb loss in people with diabetes. This is especially true in those patients with severe sepsis. Determining whether to amputate or attempt to salvage a limb often requires in depth evaluation of each individual patients physical, mental, and socioeconomic status. The current report presents and juxtaposes two similar patients, admitted to the same service at the same time with severe diabetic foot infections complicated by sepsis. We describe in detail the similarities and differences in the clinical presentation, extent of infection, etiology, and socioeconomic concerns that ultimately led to divergent clinical decisions regarding the choices of attempting diabetic limb salvage versus primary amputation and prompt rehabilitation.

N-terminal bis-(2-chloroethyl)amino and fluorosulphonyl analogues of calcitonin gene-related peptide(8-37): irreversible antagonists at calcitonin gene-related peptide receptors.

Synthesis of the first irreversible calcitonin gene‐related peptide receptor antagonists is described. bis‐(2‐Chloroethyl)amino and fluorosulphonyl groups were incorporated into the 4‐position of the N‐terminal benzoyl group of a potent competitive antagonist, N‐α‐benzoyl‐h‐α‐CGRP(8‐37) (analogues 4 and 6). Based on previous structure–activity relationships, a second pair of N‐terminally modified analogues was synthesized containing a novel benzylated‐His residue in position 10 (analogues 5 and 7). In separate experiments, SK‐N‐MC cells and mouse thoracic aortas were bathed in solutions containing 5 μm and 1.5 μm of each analogue, respectively. After extensive washing, calcitonin gene‐related peptide concentration–response curves were generated for cAMP production in SK‐N‐MC cells and relaxation of mouse aortas. All analogues caused >20% reductions in maximal calcitonin gene‐related peptide efficacy in both assays with analogue 5 containing an N‐terminal bis‐(2‐chloroethyl)amino‐benzoyl group and a benzylated‐His10 residue completely abolishing cAMP production in SK‐N‐MC cells. Reductions in maximal responses were dependent on the analogue concentration. Analogue 4 also caused more than 10‐fold reductions in the potency of the calcitonin gene‐related peptide‐mediated effects, whereas analogues 5, 6 and 7 have no significant effect on calcitonin gene‐related peptide potency. These data indicate that all analogues bind irreversibly to calcitonin gene‐related peptide receptors. The bis‐(2‐chloroethyl)amino‐modified analogues 4 and 5 were more effective than the fluorosulphonyl‐modified analogues 6 and 7.

An Efficient Synthesis of 4(5)-Benzyl-l-histidines Employing Catalytic Transfer Hydrogenolysis at Elevated Temperatures

An efficient two-step synthesis of 4(5)-benzyl-L-histidine from L-histidine was developed. A Pictet-Spengler reaction between L-histidine and benzaldehyde in the presence of excess strong base yielded 4-phenylspinacine within one hour. Catalytic transfer hydrogenolysis in methanol at reflux using ammonium formate rapidly converted 4-L-phenylspinacine to 4(5)-benzyl-L-histidine within five minutes. No racemization of the final product 4(5)-benzyl-L-histidine was observed using the Marfey reagent. To show the utility of this methodology, a series of fluorinated benzylhistidines is presented.