Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants

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

Cellular localization of relaxin-like gonad-stimulating peptide expression in Asterias rubens: New insights into neurohormonal control of spawning in starfish

Gamete maturation and spawning in starfish is triggered by a gonad-stimulating substance (GSS), which is present in extracts of the radial nerve cords. Purification of GSS from the starfish Patiria pectinifera identified GSS as a relaxin-like polypeptide, which is now known as relaxin-like gonad-stimulating peptide (RGP). Cells expressing RGP in the radial nerve cord of P. pectinifera have been visualized, but the presence of RGP-expressing cells in other parts of the starfish body has not been investigated. Here we addressed this issue in the starfish Asterias rubens. An A. rubens RGP (AruRGP) precursor cDNA was sequenced and the A-chain and B-chain that form AruRGP were detected in A. rubens radial nerve cord extracts using mass spectrometry. Comparison of the bioactivity of AruRGP and P. pectinifera RGP (PpeRGP) revealed that both polypeptides induce oocyte maturation and ovulation in A. rubens ovarian fragments, but AruRGP is more potent than PpeRGP. Analysis of the expression of AruRGP in A. rubens using mRNA in situ hybridization revealed cells expressing RGP in the radial nerve cords, circumoral nerve ring and tube feet. Furthermore, a band of RGP-expressing cells was identified in the body wall epithelium lining the cavity that surrounds the sensory terminal tentacle and optic cushion at the tips of the arms. Discovery of these RGP-expressing cells closely associated with sensory organs in the arm tips is an important finding because these cells are candidate physiological mediators for hormonal control of starfish spawning in response to environmental cues. This article is protected by copyright. All rights reserved

Development of a potentiometric flow cell with a stainless steel electrode for pH measurements. Determination of acid mixtures using flow injection analysis

In this work a stainless steel electrode was prepared, characterised and used as indicator electrode in a potentiometric flow cell. Due to its versatility, it was possible to study different electrode geometries and flow cell arrangements. After optimising the system, mixtures of succinic and oxalic acids were determined by titration. Partial least squares (PLS) regression as multivariate calibration tool was applied for data treatment. The predicted results obtained in a test set showed a relative error of 4.3% for succinic acid and 5.5% for oxalic acid. (C) 2000 Elsevier Science B.V. All rights reserved.5161163116

Artificial neural networks applied to potentiometric acid-base flow injection titrations

Artificial neural network (ANN) was applied for data treatment as a multivariate calibration tool in a potentiometric acid-base flow injection titration. A multilayer feed-forward ANN model, with Levenberg-Marquardt weight error correction was used for data modeling. The neural network parameter architecture was optimized to establish a relationship between the titration profile and the acid concentration. Citric and malic acids in synthetic sample mixtures and in orange juices were analyzed and the performance of ANN was compared with that of partial least squares (PLS) regression. (C) 2002 Elsevier Science B.V. All rights reserved.621172

Water solubilization of ethanol and BTEX from gasoline: on-line monitoring by membrane introduction mass spectrometry

The solubilization of ethanol and the aromatic hydrocarbons benzene, toluene and the isomeric ethylbenzene and xylenes (BTEX) in water in contact with commercial samples of ethanol-containing Brazilian gasoline was monitored on-line by membrane introduction mass spectrometry (MIMS) and BTEX and ethanol concentrations in water after prolonged contact were measured by flow injection analysis (FIA) coupled with MIMS (FIA-MIMS). The cosolvent effect of ethanol, which is known to increase BTEX solubility in water, was also found to speed up BTEX solubilization as compared with ethanol-free gasoline. The ethanol cosolvent effect was also compared with that of methyl tert-butyl ether (MTBE). Chemometric multivariate analysis applied to FIA-MIMS mass spectra of the ethanol-BTEX aqueous solutions formed after prolonged contact with gasoline samples showed that identical samples cluster closely whereas small variances in BTEX composition occurring among and within the three different types of ethanol-containing Brazilian gasoline are easily detected as they cause great dispersion in the chemometric plots.127223023

Quantitation Of isomeric ethyl pyridine mixtures by multivariate calibration applied to ion-molecule reaction/collision-induced dissociation triple-stage mass spectra

In reactions of the distonic ion +CH2-O-CH2. With the three isomeric ethyl pyridines, ionized methylene transfer occurs readily yielding distonic N-methylene-ethylpyridinium ions. Online mass selection and 10 eV collision-induced dissociation (CID) of the CH2+. transfer products yields characteristic fragment ions, which are formed via processes greatly influenced by the ortho, meta or para location of the ethyl substituent in the pyridine ring. Quantitation of mixtures of isomeric 2-, 3-, and 4-ethyl pyridines of varying compositions was then performed by multivariate calibration in the form of the partial least square (PLS) model applied to both single-stage (MS) 70 eV electron ionization (EI) and pentaquadrupole triple-stage sequential ion-molecule reaction/CID product ion mass spectra. The results exemplify the superior ability of combined chemometric analysis and sequential mass spectrometric techniques, which benefits from both characteristic ion chemical reactivity and dissociation behavior, for rapid and accurate quantitation of complex isomeric mixtures. (C) 2003 Elsevier Science B.V. All rights reserved.601374

Molecular identification and cellular localisation of a corticotropin-releasing hormone type neuropeptide in an echinoderm

Background: Corticotropin-releasing hormone (CRH) mediates physiological responses to stressors in mammals by triggering pituitary secretion of adrenocorticotropic hormone, which stimulates adrenal release of cortisol. CRH belongs to a family of related neuropeptides that include sauvagine, urotensin-I and urocortins in vertebrates and the diuretic hormone DH44 in insects, indicating that the evolutionary origin of this neuropeptide family can be traced to the common ancestor of the Bilateria. However, little is known about CRH-type neuropeptides in deuterostome invertebrates. Methods: Here we used mass spectrometry, mRNA in situ hybridization and immunohistochemistry to investigate the structure and expression of a CRH-type neuropeptide (ArCRH) in the starfish Asterias rubens (phylum Echinodermata). Results: ArCRH is a 40-residue peptide with N-terminal pyroglutamylation and C-terminal amidation and it has a widespread pattern of expression in A. rubens. In the central nervous system comprising the circumoral nerve ring and five radial nerve cords, ArCRH-expressing cells and fibres were revealed in both the ectoneural region and the hyponeural region, which contains the cell bodies of motoneurons. Accordingly, ArCRH-immunoreactivity was detected in innervation of the ampulla and podium of locomotory organs (tube feet), and ArCRH is the first neuropeptide to be identified as a marker for nerve fibres located in the muscle layer of these organs. ArCRH-immunoreactivity was also revealed in protractile organs that mediate gas exchange (papulae), the apical muscle and the digestive system. Conclusions: Our findings provide the first insights into CRH-type neuropeptide expression and function in the unique context of the pentaradially symmetrical body plan of an echinoderm

Pedal peptide/orcokinin-type neuropeptide signaling in a deuterostome: The anatomy and pharmacology of starfish myorelaxant peptide in Asterias rubens

Biotechnology and Biological Sciences Research Council. Grant Numbers: BB/M001644/1, BB/M001032/1; China Scholarship Counci

Direct sampling tandem mass spectrometry (MS/MS) and multiway calibration for isomer quantitation

Direct sampling tandem mass spectrometry (MS/MS) was used for the quantitation of mixtures of the isomers 2-, 3- and 4-ethyl pyridine. The similarity between the analytes and the second-order nature of MS/MS data require the use of multivariate calibration techniques capable of handling multiway data. Multilinear PLS (N-PLS) was applied here, as well as the alternative technique of unfolding the data and using standard two-way PLS. Particular attention was paid to the optimal type of spectral preprocessing. Due to the presence of heteroscedastic noise the logarithmic transform of the spectra prior to calibration gives the best results. Predictions errors of the order of 10-15% were obtained, which compare well with other results found in the literature.12781054106