Emiko Furuta

Cloning and sequencing of three C-type lectins from body surface mucus of the land slug, Incilaria fruhstorferi

Three C-type lectins of 15 kDa were isolated from the water-soluble fraction (WSF) of the body surface mucus of the land slug, Incilaria fruhstorferi. Based on their partial amino acid sequences, the nucleotide sequences of cDNAs encoding these lectins, named incilarin A, B and C, were determined. cDNAs of incilarin A, B and C consisted of 673, 663 and 715 bp, and deduced amino acids were 150, 149 and 156 residues, respectively. All three lectins had signal peptides of 17 amino acid residues at their N-termini. They showed 44-55% amino acid sequence identity with each other, and lower but significant homology with the other animal C-type lectins and antifreeze protein. Incilarin A and B seem to possess two intramolecular disulfide bonds in the carbohydrate-binding domain (CRD) conserved among the animal C-type lectins, however, one of these bonds is absent in incilarin C.

Chronic Skin Allograft Rejection in Terrestrial Slugs

Abstract To know whether or not molluscs are capable of recognizing tissue alloantigens, dorsal skin-allografts were exchanged between adult terrestrial slug, Incilaria fruhstorferi. We succeeded for the first time in orthotopic transplantation of allografts and observed chronic rejection of allografts. During the first two weeks after transplantation (WAT), in all grafts, both foreign (allo-) and self (auto-), many macrophages infiltrated from the host toward the grafts. This phenomenon is seemed to heal wounds. In the case of autografts, many macrophages observed in the grafted site until 8 weeks, whereas at 4 WAT, grafted tissues such as muscle fibers and mucous cells begun to regenerate slowly and the regeneration of these cells had been over at 20 WAT. However, in the case of allografts, regenerative phenomena were not observed, rather than muscle fibers had been actively attacked by macrophages. Numerous macrophages which phagocytosed cell debris were observed in host connective tissues during this experiment. These observations strongly suggest that an allorecognition system is present in molluscs, and in the case of terrestrial slugs dorsal skin transplantation is a useful assay system for analyses of immunological incompatibility.

Comparative studies on the internal defense system of schistosome-resistant and -susceptible amphibious snail Oncomelania nosophora 1. Comparative morphological and functional studies on hemocytes from both snails.

Abstract Two morphologically distinct blood cell types (hemocytes), Type I and Type II were found coexisting in hemolymph from two kinds of snails, Oncomelania nosophora strain, viz. from the Nirasaki strain (schistosome-resistant snail) and the Kisarazu strain (schistosome-susceptible snail). Ten min after inoculation of SRBC, the majority of Type I cells from Nirasaki strain flattened and spread over the surface of the glass plate by extending pseudopodia. In the Kisarazu strain, Type I cells adhered to the surface of substrate with spike-like filopodia, but did not form spreading lamellipodia. Type I cell from the Nirasaki strain phagocytosed SRBC but that from the Kisarazu strain did not. The starting time of recognition of foreign materials was slightly different in the Type I hemocytes from the two strains. Type II cells from both strains were round and lymphocyte-like. Ten or sixty min after incubation, Type II cells from neither strain adhered to the surface of substrate or SRBC, and did not phagocytose SRBC. Type II cells from the Nirasaki strain were quite similar to those from the Kisarazu strain. We concluded that Type I cells from the schistosome-resistant snail, Nirasaki strain, possessed higher phagocytic activity than those from the susceptible snail, Kisarazu strain, despite the morphological similarities of the hemocytes from both strains.

Perforin-Dependent Cell Death in Skin Allograft Rejection of the Terrestrial Slug Incilaria fruhstorferi

Abstract The rejection of allografts in mammals is mainly mediated by cytotoxic T-lymphocytes, whereas no comparable immunoreactive cells have been described in invertebrates. The present study was undertaken to determine whether similar cytotoxic effector cells are present when allograft rejection occurs in the terrestrial slug Incilaria fruhstorferi. A piece of dorsal skin from a donor animal was orthotopically transplanted to a recipient. Immunohistochemistry for perforin, detection of apoptosis by the TUNEL (TdT-mediated dUTP-biotin nick-end labeling) method, and electron microscopy were performed using both donor and recipient tissues. Cellular changes in the rejection process continued over for 40 days. Two functional types of “effector” cells were recognized at the rejection site, but they were observed to be macrophages possessing perforin granules and phagocytosing damaged cells of the allograft. Three days after transplantation, the perforin-positive cells were recognized only in the recipient tissue surrounding the allograft. Five days after transplantation, these cells started to appear in the graft, while they disappeared from the host tissue. However, TUNEL-positive cells were not observed throughout the graft-rejection process. Electron microscopic examination of the graft tissue revealed autophagic degeneration of epithelial cells, mucous cells, pigment cells, fibroblasts, and muscle cells. These observations suggest that the molluscan slug has the capability to recognize differences in cell-surface molecules between the allogeneic and recipient tissues, and that an allograft is chronically rejected due to a type of immunocyte that can induce perforin-dependent cell death.

Histochemical and Ultrastructural Analyses of the Epithelial Cells of the Body Surface Skin from the Terrestrial Slug, Incilaria fruhstorferi

Abstract Dorsal and ventral epithelium of the terrestrial slug, Incilaria fruhstorferi, is simple and consists of five cell types: microvillous, ciliated, round mucous, tubular mucous and channel. Microvillous cells were similar to human intestinal epithelial cells morphologically and functionally. At the base of microvilli, pinocytic vesicles which ultimately fused to form larger vacuoles, or multivesicular bodies were present. At the edge of tail or mouth, ciliated epithelial cells possessed the typical axonemes (9 plus 2 arrangement of microtubles). Mucous secretory cells were either tubular or round and their granules were membrane-bound and secreted by exocytosis. Granules of round mucous cells were proteinaceous but those of tubular cells were acidic mucopolysaccharides. Channel cells were elongate U-shaped and the central lumen was filled with a large amount of fluid (hemolymph). The function of channel cells is thought to remove hemolymph accumulated during hyperhydration. Our experiments of some markers-injection revealed that the fluid containing large molecules passed transcellularly from the hemolymph, across the basal or side region of the cell and into the central lumen. These results suggest that channel cell of the slug skin and vertebrate nephron showed some parallels in structure and function.