ECB-ART-46428
Front Neurosci
2018 Jun 08;12:382. doi: 10.3389/fnins.2018.00382.
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Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants.
Cai W
,
Kim CH
,
Go HJ
,
Egertová M
,
Zampronio CG
,
Jones AM
,
Park NG
,
Elphick MR
.
Abstract
Calcitonin (CT) is a peptide hormone released by the thyroid gland that regulates blood Ca2+ levels in mammals. The CT gene is alternatively spliced, with one transcript encoding CT and another transcript encoding the CT-like neuropeptide calcitonin-gene related peptide (α-CGRP), which is a powerful vasodilator. Other CT-related peptides in vertebrates include adrenomedullin, amylin, and intermedin, which also act as smooth muscle relaxants. The evolutionary origin of CT-type peptides has been traced to the bilaterian common ancestor of protostomes and deuterostomes and a CT-like peptide (DH31) has been identified as a diuretic hormone in some insect species. However, little is known about the physiological roles of CT-type peptides in other invertebrates. Here we characterized a CT-type neuropeptide in a deuterostomian invertebrate-the starfish Asterias rubens (Phylum Echinodermata). A CT-type precursor cDNA (ArCTP) was sequenced and the predicted structure of the peptide (ArCT) derived from ArCTP was confirmed using mass spectrometry. The distribution of ArCTP mRNA and the ArCT peptide was investigated using in situ hybridization and immunohistochemistry, respectively, revealing stained cells/processes in the nervous system, digestive system, and muscular organs, including the apical muscle and tube feet. Investigation of the effects of synthetic ArCT on in vitro preparations of the apical muscle and tube feet revealed that it acts as a relaxant, causing dose-dependent reversal of acetylcholine-induced contraction. Furthermore, a muscle relaxant present in whole-animal extracts of another starfish species, Patiria pectinifera, was identified as an ortholog of ArCT and named PpCT. Consistent with the expression pattern of ArCTP in A. rubens, RT-qPCR revealed that in P. pectinifera the PpCT precursor transcript is more abundant in the radial nerve cords than in other tissues/organs analyzed. In conclusion, our findings indicate that the physiological action of CT-related peptides as muscle relaxants in vertebrates may reflect an evolutionarily ancient role of CT-type neuropeptides that can be traced back to the common ancestor of deuterostomes.
PubMed ID: 29937709
PMC ID: PMC6002491
Article link: Front Neurosci
Genes referenced: LOC100887844 LOC115919910 LOC590297 pelp1
Article Images: [+] show captions
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Figure 1. Comparison of ArCT and ArCTP with calcitonin-related peptides and precursors from other species. (A) Neighbor joining tree showing relationships of ArCTP with CT-type precursors from other species. Reflecting phylogenetic relationships, the tree comprises a deuterostomian clade (green) and a protostomian clade (blue) and ArCTP is positioned in a branch of the deuterostomian clade that includes CT-type precursors from other ambulacrariansâthe sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) and the acorn worm Saccoglossus kowalevskii (phylum Hemichordata). The scale bar indicates amino acid substitutions per site. (B) Comparison of the sequence of ArCT with CT-related peptides from other deuterostomes (green) and from protostomes (blue). A conserved feature of all of the peptides, except CGRP-type peptides, is an amidated C-terminal proline. With the exception of protostomian DH31-type peptides, all of the peptides have a pair of cysteine residues in the N-terminal region, which have been shown to form a disulfide bond in ArCT and in other CT-type peptides. Other residues that are conserved across many of the peptides are shown with white lettering highlighted in gray. Another conserved feature is a core region of the peptides (underlined) that is predicted to form an amphipathic α-helix. The full species names, accession numbers and/or citations for the sequences included in this figure are listed in Supplemental Table 1. |
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Figure 2. Localization of ArCTP mRNA in the radial nerve cords and circumoral nerve ring of A. rubens using in situ hybridization. (a) Transverse section of a radial nerve cord that was incubated with ArCTP antisense probes showing stained cells in both the ectoneural (arrowheads) and hyponeural (arrow) regions. In the ectoneural region stained cells are concentrated laterally in the sub-cuticular epithelium, whereas in the hyponeural region stained cells are more uniformly distributed. Higher magnification images of the boxed regions are shown in panels (b,c). The inset shows an absence of stained cells in a transverse section of radial nerve cord incubated with ArCTP sense probes, demonstrating the specificity of staining observed with ArCTP antisense probes. (b) High magnification image of the apex of the V-shaped radial nerve cord, where stained cells are sparsely distributed in the sub-cuticular epithelial layer of the ectoneural region (arrowheads). (c) High magnification image of the lateral region of the radial nerve cord showing stained cells in both the ectoneural region (arrowheads) and the hyponeural region (arrows). (d) Longitudinal parasagittal section of a radial nerve cord showing that stained cells are evenly distributed along its length in the ectoneural region (arrowheads). In the hyponeural region stained cells are in segmental clusters (arrows), with each segment of the hyponeural region bounded by transverse hemal strands. Higher magnification images of the boxed regions are shown in (e,f). (e) Stained cells in the ectoneural (arrowheads) and hyponeural (arrows) regions of the radial nerve cord. (f) Junction of the hyponeural region of the radial nerve cord and a transverse hemal strand, showing that the density of stained cells is lower in this region than in the adjacent region (arrows). (g) Transverse section of the central disk region showing stained cells in both the hyponeural (arrows) and ectoneural (arrowheads) regions of the circumoral nerve ring. As in the radial nerve cords, stained cells are concentrated laterally in the ectoneural region. A high magnification image of the boxed region is displayed in panel (h), which shows stained cells in the ectoneural (arrowheads) and hyponeural (arrows) regions of the circumoral nerve ring. CONR, Circumoral nerve ring; Ec, Ectoneural region of radial nerve cord; Hy, Hyponeural region of radial nerve cord; PM, Peristomial membrane; RHS, Radial hemal strand; RNC, Radial nerve cord; TF, Tube foot; THS, Transverse hemal strand. Scale bar: 100 μm in (a,d,g); 50 μm in (A inset); 25 μm in (b,c,e,f,h). |
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Figure 3. Localization of ArCTP mRNA in the peristomial membrane and stomach of A. rubens using in situ hybridization. (a) Transverse section through the central disk region showing staining in the peristomial membrane and in the circumoral nerve ring that surrounds the peristomial membrane. High magnification images of the boxed regions are shown in (b,c). (b) Stained cells (arrowheads) in the external epithelium of the peristomial membrane. (c) Junction between the peristomial membrane and the circumoral nerve ring, with stained cells (arrowheads) in the external epithelium of the peristomial membrane and in the ectoneural epithelial layer of the circumoral nerve ring. (d) Stained cells in a section of cardiac stomach at the junction between an arm and the central disk, where the cardiac stomach is attached via nodules to extrinsic retractor strands. The boxed region is shown at higher magnification in (e), where stained cells (arrows) can be seen to be located in the basi-epithelial nerve plexus layer. (f) Transverse section through the central disk region showing stained cells in both the cardiac stomach and pyloric stomach. The boxed region in (f) is shown at a higher magnification in (g), where the stained cells (arrows) are located in the basi-epithelial nerve plexus layer. BNP, Basi-epithelial nerve plexus layer; CE, Coelomic epithelium; CONR, Circumoral nerve ring; CS, Cardiac stomach; CT, Collagenous tissue; Ec, Ectoneural region of radial nerve cord; No, Nodule; PM, Peristomial membrane; PS, Pyloric stomach; TF, Tube foot; VML, Visceral muscle layer. Scale bar: 250 μm in (a); 100 μm in (d,f); 25 μm in (b,c,e,g). |
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Figure 4. Localization of ArCTP mRNA in the pyloric ducts, pyloric caeca and rectal caeca of A. rubens using in situ hybridization. (a,b) Transverse section of a pyloric duct showing stained cells located on the oral side. The boxed region in (a) is shown at higher magnification in (b), where stained cells (arrows) can be seen to be located in the basi-epithelial nerve plexus layer. (c,d) Transverse section of an arm showing stained cells in a pyloric caecum diverticulum. The boxed region in (C) is shown at higher magnification in (d), where the stained cells (arrows) can be seen to be located in the basi-epithelial nerve plexus layer. (e,f) Transverse section of the central disk region showing stained cells in the rectum. The boxed region in (e) is shown at higher magnification in (f), where stained cells can be seen to be located in the coelomic epithelial lining of the rectum (arrows) and the body wall (arrowheads). BNP, basi-epithelial nerve plexus layer; CE, Coelomic epithelium; PC, Pyloric caecum; PD, Pyloric duct; Lu, Lumen. Scale bar: 100 μm in (a,c,e), 25 μm in (b,d,f). |
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Figure 5. Localization of ArCTP mRNA in body wall-associated tissues and organs in A. rubens using in situ hybridization. (a) Transverse section of an arm showing staining in the apical muscle. The boxed region is shown at higher magnification in (b), where stained cells (arrows) are located in the coelomic epithelial lining of the apical muscle. (c) Sagittal section of an arm showing that staining is present along the length of the apical muscle. The boxed region is shown at higher magnification in (d), where stained cells (arrows) are located in the coelomic epithelial lining of the apical muscle. (eâh) A series of transverse sections of an arm showing staining in the gonoduct that links the gonad to the gonopore in the body wall. Stained cells (arrows) are present in the coelomic epithelium of the gonoduct and at the junction between the gonoduct and the gonad. CT, Connective tissue; Go, Gonad; Gd, Gonoduct. Scale bar: 100 μm in (a,c); 25 μm in (b,d); 50 μm in (e,f,g,h). |
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Figure 6. Immunohistochemical localization of ArCT in the radial nerve cords, circumoral nerve ring and marginal nerve of A. rubens. (a) Horizontal section of a juvenile specimen showing the presence of immunostaining in the circumoral nerve ring, radial nerve cords and marginal nerves. (b) Immunostained transverse section of the V-shaped radial nerve cord showing the presence of ArCT-immunoreactive cells in both the ectoneural (arrowheads) and hyponeural (arrows) regions. In the ectoneural epithelial layer the stained cells are concentrated laterally and no stained cells are present in apical region. The inset shows an absence of staining in sections of radial nerve cord incubated with antiserum that was pre-absorbed with the ArCT peptide antigen, demonstrating the specificity of immunostaining observed in sections incubated with the antiserum. The boxed regions are displayed at high magnification in (câe). (c) Immunostained bipolar cells in the ectoneural epithelium with stained processes (arrowheads) projecting into the underlying stained neuropile (*). (d) Immunostained monopolar cells in the hyponeural region with stained processes (arrows), that run parallel to the collagenous tissue layer between the hyponeural and ectoneural regions. (e) Apical region of a radial nerve cord showing absence of stained cells in the ectoneural epithelium (arrowhead), with sparse staining of fibers in the underlying neuropile (arrow). Denser staining is present in the inner region of ectoneural neuropile (asterisk). (f) Immunostaining at the junction between a radial nerve cord and an adjacent tube foot. Immunostained processes (arrowhead) project from the ectoneural neuropile into the basi-epithelial nerve plexus of the tube foot. Immunostained processes (arrow) derived from the hyponeural region project around the margin of the peri-hemal canal to innervate the transverse infra-ambulacral muscle. (g) Immunostaining in a longitudinal section of the circumoral nerve ring showing immunostained cells in the ectoneural epithelium (arrowheads) and in the hyponeural region (arrows). Intense staining is present throughout the ectoneural neuropile. (h) Immunostaining in the marginal nerve and in the basi-epithelial nerve plexus (arrowheads) of an adjacent tube foot. Immunostained processes of the lateral motor nerve can also be seen here (arrows). CONR, Circumoral nerve ring; Ec, Ectoneural region of radial nerve cord; Es, Esophagus; Hy, Hyponeural region of radial nerve cord; MN, Marginal nerve; OS, Ossicle; RHS, Radial hemal strand; RNC, Radial nerve cord; TF, Tube foot; TIM, Transverse infra-ambulacral muscle. Scale bar: 250 μm in (a); 100 μm in (b); 50 μm in (b) inset; 10 μm in (câe); 50 μm in (f,h); 25 μm in (g). |
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Figure 7. Immunohistochemical localization of ArCT in tube feet of A. rubens. (a) Tranverse section of an arm showing the presence of immunostaining in the radial nerve cord and adjacent tube feet. Immunostaining is present in the sub-epithelial nerve plexus along the length of the stem of each tube foot and extends into the basal nerve ring in the tube foot disk. The boxed areas are shown at high magnification in (b,c). (b) Immunostaining in the sub-epithelial nerve plexus in the stem of a tube foot. (c) Immunostaining in the basal nerve ring of a tube foot disk. (d) Immunostaining in the ampulla of a tube foot. The boxed region is shown at higher magnification in (e), which shows that the immunostaining is present in fibers located beneath the coelomic lining of the ampulla. Amp, Ampulla; BNR, Basal nerve ring; CT, Collagenous tissue; ML, Muscle layer; OS, Ossicle; RNC, Radial nerve cord; TF, Tube foot. Scale bar: 250 μm in (a); 200 μm in (d); 50 μm in (b,c,e). |
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Figure 8. Immunohistochemical localization of ArCT in the peristomial membrane, esophagus and cardiac stomach of A. rubens. (a) Transverse section of the central disk region showing immunostaining in the radial nerve cords, circumoral nerve ring and peristomial membrane. The boxed region is displayed at higher magnification in (b), which shows immunostained cells (arrowheads) in the external epithelium of the peristomial membrane and immunostained fibers in the underlying basi-epithelial nerve plexus. (c) Horizontal section of the central disk region showing immunostained fibers in the esophagus. The inset shows the esophagus at high magnification, with a stained cell in the mucosal layer (arrowhead) and staining in the underlying basi-epithelial nerve plexus. (d) Transverse section of the central disk region showing both the cardiac stomach and pyloric stomach. Note that there is variation in the density of immunostaining in the basi-epithelial nerve plexus in the folds of the cardiac stomach wall, as exemplified by the two boxed areas that are shown at high magnification in (e,f). (e) Region of the cardiac stomach wall where it is attached to a nodule, which links the cardiac stomach to extrinsic retractor strands; here stained bipolar cells can be seen in the mucosal wall and the prominent basi-epithelial nerve plexus is intensely stained (********). Immunostained fibers can also be seen in the nodule. (f) Region of the cardiac stomach where stained cells are sparsely distributed and the underlying basi-epithelial nerve plexus is less intensely stained (â¦â¦â¦.) than in the adjacent region shown in (e). (g) Horizontal section of the central disk region of a juvenile starfish showing immunostaining in the cardiac stomach. The boxed region is displayed at higher magnification in (h), which shows variation in the intensity of immunostaining, with a folded region containing stained cells and a thickened and intensely stained basi-epithelial nerve plexus (********) and an adjacent region without stained cells and a less prominently stained basi-epithelial nerve plexus (â¦â¦.). BNP, Basi-epithelial nerve plexus; CS, Cardiac stomach; CONR, Circumoral nerve ring; CE, Coelomic epithelium; CT, Collagenous tissue; Es, Esophagus; Lu, Lumen; Mu, Mucosa; No, Nodule; PM, Peristomial membrane; PS, Pyloric stomach; RNC, Radial nerve cord. Scale bar: 250 μm in (a); 25 μm in (b,c) inset; 200 μm in (d,g); 100 μm in (c); 50 μm in (e,f,h). |
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Figure 9. Immunohistochemical localization of ArCT in the pyloric stomach, pyloric ducts, pyloric caeca, rectum, and rectal caeca of A. rubens. (a) Horizontal section of a juvenile specimen showing the presence of immunostaining in the pyloric stomach, pyloric ducts, and pyloric caeca. High magnification images of the boxed regions are shown in (bâd). (b) Immunostained cells (arrowheads) in the mucosa of the pyloric stomach with associated immunostained fibers in the underlying basi-epithelial nerve plexus. (c) Horizontal section of a pyloric duct showing immunostained cells (arrowheads) in the mucosa with associated immunostained fibers in the underlying basi-epithelial nerve plexus. (d) Immunostained cells (arrowheads) in the mucosa and immunostaining in the basi-epithelial nerve plexus of a pyloric caecum diverticulum. (e) Transverse section of the central disk region showing the distribution of immunostaining in the pyloric stomach. A high magnification image of the boxed region is displayed in (h), which shows an immunostained cell (arrowhead) in the mucosa and immunostained fibers in the basi-epithelial nerve plexus. (f,g) Transverse sections of a pyloric duct (f) and a pyloric caecum (g) showing immunostained fibers in the basi-epithelial nerve plexus; note that the staining in the pyloric duct is more prominent on the oral (lower) side, as shown at higher magnification in (i). (j) High magnification image of the boxed region in (g), showing immunostaining in the basi-epithelial nerve plexus of a pyloric caecum diverticulum. (k) Transverse section of the central disk region showing immunostaining in the rectal caeca and rectum. High magnification images of the boxed regions are displayed in (l,m). Immunostaining can also be seen in the inner region of the rectum (arrows) that is linked to the pyloric stomach. (l). Junction between the rectum and the anal opening in the aboral body wall of the central disk; note the instense staining in the nerve plexus (arrows) beneath the coelomic epithelium of the rectum. (m) Immunostaining in both the visceral muscle layer (arrowhead) and sub-mucosal basi-epithelial nerve plexus (arrow) of a rectal cecum. An, Anus; BNP, Basi-epithelial nerve plexus; CS, Cardiac stomach; Ce, Coelomic epithelium; Lu, Lumen; Mu, Mucosa; PC, Pyloric caecum; PD, Pyloric duct; PS, Pyloric stomach; Re, Rectum; RC, Rectal caecum. Scale bar: 250 μm in (a); 150 μm in (e,g,k); 100 μm in (f); 40 μm in (j,l,m); 25 μm in (b,c,d,h); 20 μm in (i). |
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Figure 10. Immunohistochemical localization of ArCT in body wall-associated tissues/organs in A. rubens. (a) Tranverse section of an arm showing immunostaining in the apical muscle and an adjacent papula. A high magnification image of the boxed region is shown in (b). (b) Immunostained cells (black arrowheads) are present in the coelomic lining of the apical muscle and profiles of immunostained fibers (arrows) are amongst the longitudinally orientated muscle fibers of the apical muscle. Immunostained fibers can also be seen here in the circular muscle layer of the body wall (white arrowhead). (c) Transverse section of an arm showing the presence of immunostaining in the nerve plexus beneath the coelomic epithelial lining of a papula (arrowheads), which is contiguous with the basi-epithelial nerve plexus of the epithelium lining the main coelomic cavity of the arm (arrows). Immunostaining can also be seen here in association the circular muscle layer (white arrowheads) and an immunostained process derived from this layer can be seen projecting into the body wall (white arrow). (d) Immunostained nerve fibers (arrows) associated with strands of muscle that are attached to body wall ossicles and are derived from the circular muscle layer of the body wall (e) Transverse section of the aboral body wall of an arm, showing immunostained fibers associated with interossicular muscles, with the boxed region shown at higher magnification in (f). (g) Immunostaining in the sub-epithelial nerve plexus (arrows) of the aboral body wall external epithelium. (h) Transverse section of an arm showing immunostaining (arrows) in the gonoduct that connects the gonad to the gonopore. AM, apical muscle; CBNP, Coelomic basi-epithelial nerve plexus; CML, circular muscle layer; CMLNP, Circular muscle layer nerve plexus; CT, collagenous tissue; Go, Gonad; Gd, Gonoduct; IOM, Inter-ossicular muscle; OS, Ossicle; Pa, Papula; PC, Pyloric caecum; Scale bar: 200 μm in (a,c,e); 100 μm in (g,h); 25 μm in (b,d); 10 μm in (f). |
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Figure 11. ArCT causes dose-dependent relaxation of tube foot and apical muscle preparations from A. rubens. (A,B) Representative recordings showing that synthetic ArCT causes dose-dependent relaxation of tube foot (A) and apical muscle (B) preparations that had been stimulated to contract by application of 10 μM acetylcholine (ACh). (C,D) Graphs showing the dose-dependent relaxing effect of synthetic ArCT on tube foot (C) and apical muscle (D) preparations. Mean values ± standard deviation were determined from three experiments, using preparations from different individuals. Relaxing activity was quantified as the percentage reversal of contraction induced by application of 10 μM ACh. |
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Figure 12. Purification and identification of a muscle relaxant from the starfish P. pectinifera that is a calcitonin-type peptide. (A) Fractionation of an extract of P. pectinifera by cation-exchange chromatography using a linear gradient of 0.02 to 1.5 M ammonium acetate (pH 5.0) at a flow rate of 2.75 ml/min over 6 h. Fractions 40â45 and fractions 49 and 50 exhibited relaxing activity on apical muscle preparations. The bioactive constituent of fractions 40â45 was identified previously as the pedal peptide-type neuropeptide SMP (Kim et al., 2016). The bioactive constituent of fractions 49 and 50, designated âPeak A,â was purified here. (B) Following several purification steps, cation-exchange HPLC (0â0.22M NaCl over 22 min) was used here to obtain a single absorbance peak containing bioactive peak A. The inset shows that an aliquot of purified peak A causes relaxation of an apical muscle preparation from P. pectinifera that had been contracted with 10â6 M ACh. (C) Analysis of peak A using MALDI TOF MS reveals that doubly and singly charged ions have molecular masses of 1828.9 and 3655.1 Da, respectively. (D) Partial sequence of purified peak A, where PEC represents modified cysteine caused by reduction and alkylation. (E) Determination of the sequence of peak A based upon mass spectral analysis and sequencing of the purified peptide and sequencing of a cDNA encoding its precursor (see supplementary Figure 3) reveals that it is a calcitonin-type peptide (PpCT) that shares sequence similarity with ArCT. |
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Figure 13. Quantitative analysis of the expression levels of the PpCT precursor transcript in P. pectinifera organs/tissues and pharmacological analysis of the relaxing effect of synthetic PpCT on apical muscle preparations. (A) Analysis of the expression of the PpCTP transcript using RT-qPCR reveals that the highest expression level is in radial nerve cords. Expression was also detected in other organs/tissues analyzed but at a level that is two orders of magnitude lower than in radial nerve cords. Mean values with standard deviations (n = 3) are shown. (B) Synthetic PpCT causes dose-dependent relaxation of apical muscle preparations from P. pectinifera. Mean values ± standard deviation were determined from 5 separate experiments. Relaxing activity was calculated as the percentage reversal of contraction of the apical muscle caused by 10â6 M ACh. |
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Figure 14. Schematic showing the phylogenetic distribution and structural properties of calcitonin-related peptides in the Bilateria and the occurrence of calcitonin-related peptides that have been shown to act as muscle relaxants. Calcitonin and at least five other calcitonin-related peptides typically occur in vertebrate species, whereas in deuterostomian invertebrates, there is typically only a single CT-type peptide. Several calcitonin-related peptides act as muscle relaxants in vertebrates. Our discovery that CT-type peptides act as muscle relaxants in starfish (phylum Echinodermata) indicates that the evolutionary origin of this physiological role can be traced back to the common ancestor of the deuterostomes. In the protostomian lineage gene duplication has given rise to two types of calcitonin-related peptidesâCT-like peptides with an N-terminal disulfide bond and DH31-type that lack an N-terminal disulfide bond. In protostomian invertebrates, the physiological roles of DH31-type peptides have been characterized (see discussion) but nothing is known about the physiological roles of CT-like peptides with an N-terminal disulfide bond. |
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