Occurrence and diversity of barnacles on international ships visiting Osaka Bay, Japan, and the risk of their introduction.

The occurrence and diversity of barnacles attached to the hulls of two intercontinental bulk carriers were studied at the port in Osaka Bay, Japan, to assess the potential risk of introduction of exotic species to the inner part of the bay. Barnacles were sampled from the bulbous bows, near the midship draft marks, and around the propeller posts and rudders. Twenty-two species were found, with 14 of these not previously recorded in Osaka Bay, including four species not previously recorded elsewhere in Japan. From an assessment of environmental similarity between Osaka Bay and native ranges, of the 14 species, Elminius modestus and Amphibalanus variegatus were considered to have the highest risk of introduction to the inner part of the bay.

Possible functions of oxytocin/vasopressin-superfamily peptides in annelids with special reference to reproduction and osmoregulation.

Annetocin is an earthworm oxytocin-related peptide that we previously isolated from the whole body of a lumbricid earthworm Eisenia foetida. We have reported that annetocin induces egg-laying-like behaviors in E. foetida and a gnathobdellid leech, Whitmania pigra, when it is injected into the respective animals. The present study was undertaken to probe physiological functions of invertebrate oxytocin-vasopressin-superfamily peptides with special reference to reproductive and osmoregulatory events in which vertebrate peptides of this superfamily are involved. Annetocin, Lys-conopressin (a leech vasopressin-related peptide) and two analog peptides, [Tyr(3)]-annetocin ((3)Y-annetocin) and [Phe(3)]-annetocin ((3)F-annetocin), were compared for their activities to induce egg-laying-like behavior and to change body weight as a measure of water balance in the leech W. pigra. Injection of annetocin, Lys-conopressin, and (3)F-annetocin caused both egg-laying-like behavior and reduction of body weight in the animals, but (3)Y-annetocin induced neither. Furthermore, leeches in the non-breeding season responded to peptides less conspicuously than those in the breeding season. Such a concomitant induction of egg-laying-like behavior and body-weight reduction suggests that these two phenomena are unitary and might be accounted for by the fact that egg-laying in leeches and earthworms is accompanied by secretion of a large quantity of mucus, which should significantly contribute to body-weight loss. J. Exp. Zool. 284:401-406, 1999.

Localization of leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization.

We have recently isolated a myoactive peptide, called leech excitatory peptide, belonging to the GGNG peptide family from two species of leeches, Hirudo nipponia and Whitmania pigra. Immunohistochemistry and in situ hybridization were employed to localize leech excitatory peptide-like peptide(s) and its gene expression in the central nervous system of W. pigra. A pair of neuronal somata were stained by both immunohistochemistry and in situ hybridization in the supraesophageal, subesophageal, and segmental ganglia. In addition, several other neurons showed positive signals by either immunohistochemistry or in situ hybridization in these ganglia. An immunoreactive fiber was observed to run in the anterior root of segmental ganglion 6, which is known to send axons to the sexual organs, though we failed to detect immunoreactivity in possible target tissues. Antiserum specificity was established by enzyme-linked immunosorbent assay using different leech excitatory peptide-related peptides. Leech excitatory peptide elicited muscular contraction of isolated preparations of penis and intestine at concentrations of 10(-8 )M. These results suggest that leech excitatory peptide is a neuropeptide modulating neuromuscular transmission in multiple systems, including regulation of reproductive behavior.

Comparison of precursor structures of the GGNG peptides derived from the earthworm Eisenia foetida and the leech Hirudo nipponia.

Earthworm and leech cDNAs encoding the GGNG peptides, a family of myotropic peptides, were cloned and examined in this study. Both of the predicted precursor proteins are of polyprotein structure and contain several putative peptides distinct from the GGNG peptides. However, the precursors show organizations distinct from each other and no sequence similarity except for the GGNG peptides.

Immunohistochemical Localization of Annetocin, an Earthworm Oxytocin-Related Peptide, and Identification and Ultrastructural Characteristics of the Annetocin-Secretory Cells in the Oligochaete Earthworm Eisenia foetida.

Annetocin is an egg-laying-inducing oxytocin-related peptide which we have previously isolated from the earthworm, Eisenia foetida. Here we report the results of immunohistochemical and ultrastructural studies on annetocin-secretory cells in the earthworm. Annetocin-immunoreactive (IR) cell-somata were located mainly at the ventro-lateral side of the subesophageal ganglion. Only four annetocin-IR cells were seen in the cerebral ganglion. Some annetocin-IR cells displayed unipolar-like structure with a process directing to the core region (the neuropile) of the ganglion. Annetocin-IR fibers were also observed in the neuropile of the ventral ganglia and the ventral nerve cord between the 4th and the 30th segments including the clitellum, but not in the posterior segments (31-55th). The number of annetocin-IR fibers decreased from the 4th to the 30th segment. The annetocin-secretory cells were identified by the immunogold staining, and filled with gold-labeled vesicles, 200-250 nm in diameter, which included moderately electron dense material. The annetocin-secretory cells possessed a euchromatic nucleus, well-developed rough endoplasmic reticulum and Golgi apparatus. Some of the annetocin-secretory cells were found to form a neurohemal-like structure, where somata or fibers with loose glial investment came in contact with the coelomic space at the ventral side of the subesophageal ganglion. The results suggest that annetocin is a neuropeptide produced and secreted by the neuron in the cerebral and subesophageal ganglia of the earthworm.

The leech excitatory peptide, a member of the GGNG peptide family: isolation and comparison with the earthworm GGNG peptides.

A member of the GGNG peptide family was isolated from Hirudo nipponia (leech). GGNG peptides had only been isolated previously from earthworms. The C-terminus structure of the leech peptide, LEP (leech excitatory peptide), was -Gly-Gly-Asn-amide, while that of the earthworm peptides, EEP (earthworm excitatory peptide), was -Gly-Gly-Asn-Gly. LEP exerted 1000-fold more potent activities on leech gut than did EEP-2. On the other hand, EEP-2 was 1000-fold more potent than LEP on the crop-gizzard of the earthworm. Analog peptides of LEP and EEP-2 were synthesized, and the myoactive potency of each analog on the leech and earthworm tissues was compared.

Immunochemical demonstration of Eisenia tetradecapeptide, a bioactive peptide isolated from the gut of the earthworm Eisenia foetida, in tissues of the earthworm.

The quantity and localization of Eisenia tetradecapeptide which was isolated from the gut of the earthworm Eisenia foetida were examined in tissues of the same species by enzyme-linked immunosorbent assay and immunohistochemistry. Analysis by enzyme-linked immunosorbent assay showed that Eisenia-tetradecapeptide-like immunoreactivity was present in both the central nervous system (cerebral ganglion, subesophageal ganglion, ventral ganglia, and ventral nerve cord) and the gut (esophagus, crop, gizzard, and intestine). The central nervous system contained a higher amount of Eisenia-tetradecapeptide-like immunoreactivity (1.3 pmol/mg wet weight) than the gut (0.2-0.6 pmol/mg wet weight). Eisenia-tetradecapeptide-like immunoreactivity was scarcely detected in the body-wall muscle, nephridia, and sexual organs (testis, ovary, seminal vesicle, and ovisac). Immunohistochemical analysis demonstrated that intense Eisenia-tetradecapeptide-like immunopositive cells and nerve fibers were present in the central nervous system. Immunoreactivity was found in the epithelial cells lining the esophagus and in the submucous plexus in various parts of the gut. Thus, the present study suggests that Eisenia tetradecapeptide is a neuropeptide and/or peptide hormone present in both the central nervous system and the gut of the earthworm and that its role involves the regulation of gut motility.

Annetocin, an annelid oxytocin-related peptide, induces egg-laying behavior in the earthworm, Eisenia foetida.

Annetocin, an oxytocin-related peptide which we isolated from the earthworm Eisenia foetida, induced a series of egg-laying-related behaviors in the earthworms. These stereotyped behaviors consisted of well-defined rotatory movements, characteristic body-shape changes, and mucous secretion from the clitellum. Each of these behaviors is known to be associated with formation of the cocoon in which eggs are deposited. In fact, some of the earthworms injected with annetocin (> 5 nmol) laid eggs. Such egg-laying-related behaviors except for oviposition were also induced by oxytocin, but not by Arg-vasopressin and some other bioactive peptides isolated from E. foetida. Furthermore, annetocin also induced these egg-laying-like behaviors in the leech Whitmania pigra, but not in the polychaete Perinereis vancaurica. These results suggest that annetocin plays some key role in triggering stereotyped egg-laying behaviors in terrestrial or fresh-water annelids that have the clitella.

The GGNG peptides: novel myoactive peptides isolated from the gut and the whole body of the earthworms.

Three novel bioactive peptides, which were structurally related to each other, were purified and chemically identified from two species of the earthworms, Eisenia foetida and Pheretima vittata. One peptide was isolated from the gut tissue of E. foetida, and the other two were purified from the whole body of E. foetida and P. vittata, respectively. These peptides consisted of 17 or 18 amino acid residues and were named GGNG peptides after their unique, common structure of the C-termini. These GGNG peptides augmented both tension and frequency of spontaneous contractions of isolated gut preparations of E. foetida, and also elicited contractions of other annelidan tissues such as the polychaete esophagus and the leech vagina. However, they showed no activity on molluscan or arthropodan tissues, suggesting that GGNG peptides may be peculiar to annelids. No peptides homologous to GGNG peptides have been known so far in any living organisms.

Effects of annetocin, an oxytocin-related peptide isolated from the earthworm Eisenia foetida, and some putative neurotransmitters on gut motility of the earthworm.

Annetocin, an oxytocin-related peptide recently isolated from the lumbricid earthworm Eisenia foetida, and putative transmitter substances were examined for their effects on rhythmic, spontaneous contractions of isolated gut preparations of the earthworm. Significant, dose-dependent effects of the following substances were observed: acetylcholine (ACh), gamma-aminobutyric acid (GABA), and dopamine were excitatory, while serotonin (5-HT) and octopamine were inhibitory. Annetocin, oxytocin, and vasotocin stimulated spontaneous contraction of the earthworm gut, annetocin being approximately 10-fold more potent than oxytocin or vasotocin. However, arginine-vasopressin (Arg-vasopressin), lysine-vasopressin (Lys-vasopressin), tocinoic acid (N-terminal hexapeptide fragment of oxytocin), and MSH release-inhibiting factor (MIF; C-terminal tripeptide fragment of oxytocin) did not show any effect on the earthworm gut motility. On the other hand, oxytocin, vasotocin, Arg-vasopressin, Lys-vasopressin, and tocinoic acid caused spontaneous contractions of isolated rat uterine preparations, where the potency was in this order, while annetocin and MIF exerted no oxytocic activity on the uterus. Dose-response relationship of the effects of annetocin and its related peptides on the annelid and mammalian systems shows that amino acid residue at the third position of these peptides is important for exertion of excitatory action on the smooth muscle systems. The results in the present study suggest that receptors for annetocin and for GABA on the earthworm gut, unlike those for ACh, desensitize during continuous exposure to these substances.

A novel gut tetradecapeptide isolated from the earthworm, Eisenia foetida.

A novel bioactive tetradecapeptide, GFKDGAADRISHGFamide, was isolated from the gut of the oligochaete annelid, Eisenia foetida, using the isolated anterior gut (crop-gizzard) as a bioassay system. A highly homologous peptide, GFRDGSADRISHGFamide, was also purified from the whole body of another species of earthworm, Pheretima vittata. These peptides were termed Eisenia tetradecapeptide (ETP) and Pheretima tetradecapeptide (PTP), respectively. Both the peptides showed a potent excitatory action on spontaneous contractions of the anterior gut with a threshold as low as 10(-10)-10(-9) M. These peptides were significantly homologous to molluscan tetradecapeptides and, to a lesser extent, to arthropodan tridecapeptides that have been reported to date. All these peptides seem to be evolutionally related to each other.

Bioactive peptides of annelids are closely related to those of molluscs.

Several bioactive peptides were isolated from some annelids. Each of the annelidan peptides seemed to be a member of a previously found molluscan peptide family.

Annetocin: an oxytocin-related peptide isolated from the earthworm, Eisenia foetida.

An oxytocin-vasopressin-related peptide, Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2, was isolated from the lumbricid earthworm, Eisenia foetida and termed annectocin. Annetocin potentiated not only spontaneous contractions of the gut but also pulsatory contractions and bladder-shaking movement of the nephridia. Annetocin may be involved in osmoregulation of the animal through nephridial function.

[Clinical experiences with intraluminal irradiation using 198Au grains in the treatment of bile duct carcinoma].

Three cases of a bile duct carcinoma were treatment with radiotherapy, using intracatheterized 198Au grains. The intracatheter 198Au grains, placed in an inner tube, were inserted into a percutaneous transhepatic catheter. The number of 198Au grains used depended upon the length of the tumor. Tips of plastic were placed between the grains to improve the spatial and temporal dose allocation. The number of grains used can be changed quite easily, according to the length of the tumor, and the period of insertion in our cases was shorter than for a 192Ir wire. These three patients also received external irradiation and, since completion of treatment, two have continued to do well for the past 5 months. There have been no systemic or local complication.