Cionin: a disulfotyrosyl hybrid of cholecystokinin and gastrin from the neural ganglion of the protochordate Ciona intestinalis.

We have purified an acidic octapeptide from the neural ganglion of the protochordate Ciona intestinalis by a three-step procedure including C18 Sep-Pak fractionation, MonoQ ion-exchange chromatography, and C4 reversed-phase high-performance liquid chromatography. The purification was monitored by an immunoassay specific for the alpha-carboxyamidated COOH terminus common to the mammalian brain-gut hormones, cholecystokinin and gastrin. Automated Edman degradation revealed the sequence Asn-Tyr-Tyr-Gly-Trp-Met-Asp-Phe. In accordance with the high acidity of the peptide, amino acid analysis after cleavage with aminopeptidase M showed that both tyrosyl residues are sulfated. Hence, the structure is Asn-Tyr(SO3)-Tyr(SO3)-Gly-Trp-Met-Asp-Phe-NH2, as also confirmed by identity with the synthetic disulfated peptide in different chromatographic systems. The occurrence of two consecutively sulfated tyrosyl residues after a neutral residue challenges present concepts of consensus sites for tyrosyl sulfation. We conclude that the structure of the peptide, named cionin, suits that of a common ancestor for cholecystokinin and gastrin.

Distribution of ion channels in ascidian eggs and zygotes.

Ascidian eggs and zygotes were whole-cell voltage-clamped and inward membrane currents, generated by stepping the membrane potential, studied from fertilization up to cytokinesis. Currents, induced by changing the voltage in steps from -80 to -30 mV, or to 0 mV, had maximum amplitudes which ranged from 400 to 1200 pA in the unfertilized egg and 100 to 1300 pA in the zygote. At 5 to 10 min after fertilization it was not possible to generate inward currents owing to the activity of nonspecific fertilization channels. Preceding cytokinesis, we observed a reduction in amplitude of the inward currents. By cutting eggs and zygotes into fragments, we have shown that the ion channels generating these inward currents are symmetrically distributed over the egg plasma membrane, but regionalized in the zygote with a maximum density at the animal pole.

Sulphated CCK-8-like peptides in the neural ganglion of the protochordate Ciona intestinalis.

Immunochemical studies were carried out on extracts of the neural ganglion from the ascidian Ciona intestinalis in order to the characterize the peptide(s), which react with antibodies against the C-terminal sequence common for the mammalian hormones, cholecystokinin (CCK) and gastrin. Radioimmunoassays specific for the sulphotyrosyl-containing N-terminus of CCK-8, for the common alpha-carboxyamidated C-terminus and for gastrin were used to monitor gel chromatography and reverse-phase HPLC of the extracts. Only neutral extracts contained immunoreactive material (634 (524-785) pmol eqv.CCK-8/g) (mean and range, n = 4)). HPLC revealed a small peak eluting almost like CCK-8 and a larger peak eluting earlier. By subsequent gel chromatography the larger peak eluted in the same position as sulphated CCK-8. The material was recognized almost equally by the N- and C-terminal CCK radioimmunoassays, whereas the specific C-terminal gastrin radioimmunoassay did not measure the peptides. Treatment with arylsulphatase removed the binding to the antiserum specific for the sulphotyrosyl-containing sequence of CCK. The results indicate that the ganglion of Ciona intestinalis contains a tyrosyl-sulphated peptide resembling mammalian CCK-8.

Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis.

Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.

Gastro-intestinal and neurohormonal peptides in the alimentary tract and cerebral complex of Ciona intestinalis (Ascidiaceae).

Polypeptide-hormone producing cells were localized in the alimentary tract and cerebral ganglion of Ciona intestinalis using cytochemical, immunocytochemical and electron-microscopical methods. Antisera to the following peptides of vertebrate type were employed: bombesin, human prolactin (hPRL), bovine pancreatic polypeptide (PP), porcine secretin, motilin, vasoactive intestinal polypeptide (VIP), beta-endorphin, leu-enkephalin, met-enkephalin, neurotensin, 5-hydroxytryptamin (5-HT), cholecystokinin (CCK), human growth (GH), ACTH, corticotropin-like intermediate lobe peptide (CLIP) and gastric inhibitory peptide (GIP). Immunoreactive cells were found both in the alimentary tract epithelium and in the cerebral ganglion for bombesin, PP, substance P, somatostatin, secretin and neurotensin. Additionally, in the cerebral ganglion only, there were cells immunoreactive for beta-endorphin, VIP, motilin and human prolactin. 5-HT positive cells, however, were restricted to the alimentary tract. No immunoreactivity was obtained either in the cerebral ganglion or in the alimentary tract with antibodies to leu-enkephalin, met-enkephalin, CCK, growth hormone, ACTH, CLIP and GIP. Prolactin-immunoreactive and pancreatic polypeptide-immunoreactive cells were argyrophilic with the Grimelius' stain and were found in neighbouring positions in the cerebral ganglion. At the ultrastructural level five differently granulated cell types were distinguished in the cerebral ganglion. Granules were present in the perikarya as well as in axons. The possible functions of the peptides as neurohormones, neuroregulators and neuromodulators are discussed.

[Observation, by immunofluorescence, of an LH-RH-like antigen in the nervous system of Ciona inteesinalis (Tunicier ascidiacea)]. / Mise en évidence par les techniques d'immunofluorescence d'un antigène de type LH-RH dans le système nerveux de Ciona intestinalis (Tunicier ascidiacé).

Using Rabbit anti-LH-RH antiserum for immunofluorescence, the presence of an LH-RH-like antigen was observed in the nervous system of a Protochordate Ciona intestinalis. Few immunoreactive cells are localized on the ventral posterior part between the nervous ganglion and the neural gland. Another group of cells are visible along the small nervous fibers near the dorsal vessel.

Substance P-, neurotensin- and bombesin-like immunoreactivities in the gill epithelium of Ciona intestinalis L.

Substance P-, neurotensin- and bombesin-like immunoreactivities were localised in some gill epithelial cells in the pharynx of Ciona intestinalis L. No immunoreactivity was obtained with antisera to gastrin, glucagon, insulin, pancreatic polypeptide or calcitonin. Some of the epithelial cells of the gills were shown to be argyrophilic with the Grimelius technique.

Calcitonin-like immunochemical staining in the alimentary tract of Ciona intestinalis L.

Calcitonin-like immunoreactivity has been found with the peroxidase-anti-perioxidase (PAP) method in cells of the epithelium of the alimentary tract as well as in nerve cells and nerve fibers in the connective tissue underlying the epithelium of the alimentary tract of Ciona intestinalis L. The nature of these cells is discussed with reference to endocrine-like cells found in the alimentary tract of other protochordates and to the possible dual role of calcitonin occurring in the gastroenteropancreatic system, on the one hand, and in the nervous system, on the other.

Immunological evidence for an ACTH-like antigen in the nervous ganglion of a protochordate Ciona intestinalis (Ascidiacea).

Nervous fibers and neurons containing an ACTH-like antigen could be demonstrated in the nervous ganglion of a Protochordate Ciona intestinalis, using indirect immunofluorescence and anti (17-39)-ACTH and anti (25-39)-ACTH antisera.

Erythrocyte agglutinins in the blood of certain ascidians.

Plasma from Ciona intestinalis, Phallusia mamillata and Ascidia malaca possess hemagglutinin for a variety of erythrocytes. Results obtained by physical and chemical treatments suggest that hemagglutinin for Phallusia mamillata and Ascidia malaca may be a protein or a protein-like substance.