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Allo-recognition and rejection of skin grafts in the terrestrial slug, Indiana fruhstorferi
Eighth JADCI abstracts
67
did not affect wound repair. Histological observation of cut surfaces by HE staining wound healing occurred revealed that completely 3 hrs after cutting. Then, using planaria with healed surfaces, the effect of antibodies on regeneration was studied. Results revealed that regeneration occurred in the presence of anti-HSP, but not in the autolysis was of anti-lectin; presence observed after 48 hr-exposure. These results strongly suggest that HSP60 molecule is required in wound healing and that the body surface mucus lectin is also necessary in regeneration.
chromatography and ultrafiltration of cut off 10,000. The antifungal material is a small peptide and stable after treatment at 100°C for 10 minutes, thus it differs from other dolabellanins. MORPHOLOGICAL B3. FUNCTIONAL CHARACTERIZATION HEMOCYTES IN THE CLAM, crocea. Tridacna
NOVEL FROM
Ryosuke Iijima, Jun Kisugi and Masatoshi Faculty of Pharmaceutical Yamazaki. Sciences, kimagawa
Teikyo University,
Sagamiko-cho,
199-01, Japan. Broad range antimicrobial activities have been exhibited by each group of humoral self-defense factors of invertebrates such as sarcotoxins, cecropins and insect defensins. It can be expected that the combination of these factors contributes to effective immunodefense systems against infectious microorganisms. Homologues of these small peptide factors have been observed not only in insects but also in mammals. Thus, these factors may have evolved before separation of the protostomes and the deuterostomes. We previously purified four cytotoxic proteins named dolabellanins from the sea hare Dolabella auricuralia, the shell-less opisthobranch mollusk. However, the dolabellanins are not small heat-labile proteins. Therefore, we performed experiments designed to find the antimicrobial peptides from D. auricuralia. An antifungal activity, that killed Saccharomyces cerevisiae after two hours coincubation, was extracted by 60% acetonitrile- 1% triofluoroacetate/water from skin and body wall. Further purification involved reverse-phase and cation exchange
OF GIANT
Koji Nakayama, Ann M. Nomoto, Miyuki Nishijima and Tadashi Maruyama.
Marine
Biotechnology
Laboratories,
B2. PURIFICATION OF ANTIFUNGAL ACTIVITY SEA HARE BODY WALL.
AND
Shimizu
Institute, 424,
Shimizu
Japan.
To
understand the cellular immunodefense system in the giant clam, Trdacna crocea, which harbors symbiotic microalga zooxanthella, hemocytes were characterized using light and electron microscopy. We observed of hemocytes: three types eosinophilic granulocytes, agranular cells and morula-like cells. Eosinophilic granulocytes contained granules approximately 0.6 pm in diameter. Agranular cells adhered to glass surfaces and contained electron lucent granules and few electron dense granules. Morula-like cells were packed with large (approximately 3 pm in diameter), electron dense granules. Eosinophilic granulocytes were acid phosphatase positive and were phagocytic against latex particles. Agranular cells and morula-like cells lacked phagocytic ability. When the hemolymph was exposed to seawater, hemocytes aggregated to form large clots and agranular cells were the main component found in the core of the clots. Morula-like cells released large granules during aggregation and they contained protein molecules having a molecular weight of 7.4 kDa. (Supported in a part by The Industrial Science and Technology Frontier Program by the New Energy and Industrial Technology Development Organization (Tokyo, Japan)). B4.
ALLO-RECOGNITION
AND
REJECTION OF SKIN GRAFTS IN THE TERRESTRIAL SLUG,
68
Zncilaria fruhstorferi. Yamaguchi, Emiko Hiroaki Nakamura*.
Eighth JADCI abstracts
Keiichiro Furuta* and i%e Laboratory of
fiuhstorferi
is composed chiefly of three cell types: 1) microvillous epithelial cells, 2) mucous cells, and 3) U-shaped cells. These Medical Sciences and *Department of are connected at their apical margins by Anatomy, Dokkyo University School of junctional complexes. In our ultrastructural Medicine, Mibu, Tochigi 321-02, Japan. In studies, we often observed that the U-shaped molluscs, the presence of allo-recognition has cells contained homogeneous components. not yet been proved, although heterotopic When carbon particles were injected into the transplantations have been tried. In the slug’s hemocoel, the particles passed rapidly present study, we transplanted auto- and through the skin. However, they were neither allografts of the dorsal skin to the same observed in the microvillous nor mucous Incilaria cells. We searched the route of passage of position in the land slug, fruhstotferi. Allo-rejection was judged using materials, using carbon particles, yeasts, a dissecting microscope and was characterized FITC-labeled dextran (MW 70 kDa), by disappearance of the graft outline and its hemocyanin antibodies and rhodamine-labeled shrinkage. In allografls, 76.9% of successful latex beads @=0.03p.m). Besides yeasts, grafts was rejected 12-26 days after these materials were found in tubule-like transplantation, while 62.5% of autografts spaces among the epithelial cells. Thirty min after injection of rhodamine-labeled latex survived for at least 26-44 days and statistical beads or FITC-labeled dextran, these indicated significant analysis (x2 -test) materials, which were contained in the body differences. By light microscopy, melanin surface mucus, were detected by fluorescence granules phagocytosed by macrophage-like cells were observed in allografis, but intact microscopy in the U-shaped cells. Seventy in melanocytes were observed in autografts. By hrs later, they were still distinguishable electron microscopy, a greater number of the mucus. These results revealed that the Uexogenous were found in shaped cells exudated these macrophage-like cells connective tissue of allografts when compared materials and endogenous hemocyanin (MW These materials might be to autografts and the number of mucous cells 400 kDa). markedly decreased in both alloand endocytosed and then released into the lumen autografts. Transplantation in molluscs is by exocytosis. difficult technically thus further observations and Host Defense in are essential in order to distinguish between (C) Hemocytes destroyed grafts caused by faulty technique vs Protochordates immuno-rejection. Cl. HISTOLOGICAL ANALYSIS HEMOCYTES AND OF B5. MOST SURPRISING CELLS TISSUE IN AMONG EPITHELIAL CELLS OF HEMOPOIETIC Halocynthia roretzi. Tomoo Sawada, WALL FROM THE BODY Nobuko Tokuda, Yoshihisa Fujikura Zncilaria TERRESTRIAL SLUG, and Tetsuo Fukumoto. Department of Furuta, Emiko fruhstorferi. Anatomy, Yamaguchi University School of Hiroaki Keiichiro Yamaguchi*, Medicine, Ube 755, Japan. We examined the Nakamura and Shin-ichi Kikuchi**. Department of Anatomy and *L.aboratory of distribution of various hemocytes in tissues of HaZocynthia roretzi, in order to improve Medical Science, Dokkyo University School in light-microscopic of Medicine, Mibu, Tochigi 321-02, Japan hemocyte identification and **Marine Ecosystems Research Center, tissue sections. Vacvolated cells remain inside the sinus-like spaces within connective Chiba University, Kominato, Chiba 299-55, Japan. Body wall epithelium of Incilaria tissue of the endocarp, but rarely infiltrated
67
did not affect wound repair. Histological observation of cut surfaces by HE staining wound healing occurred revealed that completely 3 hrs after cutting. Then, using planaria with healed surfaces, the effect of antibodies on regeneration was studied. Results revealed that regeneration occurred in the presence of anti-HSP, but not in the autolysis was of anti-lectin; presence observed after 48 hr-exposure. These results strongly suggest that HSP60 molecule is required in wound healing and that the body surface mucus lectin is also necessary in regeneration.
chromatography and ultrafiltration of cut off 10,000. The antifungal material is a small peptide and stable after treatment at 100°C for 10 minutes, thus it differs from other dolabellanins. MORPHOLOGICAL B3. FUNCTIONAL CHARACTERIZATION HEMOCYTES IN THE CLAM, crocea. Tridacna
NOVEL FROM
Ryosuke Iijima, Jun Kisugi and Masatoshi Faculty of Pharmaceutical Yamazaki. Sciences, kimagawa
Teikyo University,
Sagamiko-cho,
199-01, Japan. Broad range antimicrobial activities have been exhibited by each group of humoral self-defense factors of invertebrates such as sarcotoxins, cecropins and insect defensins. It can be expected that the combination of these factors contributes to effective immunodefense systems against infectious microorganisms. Homologues of these small peptide factors have been observed not only in insects but also in mammals. Thus, these factors may have evolved before separation of the protostomes and the deuterostomes. We previously purified four cytotoxic proteins named dolabellanins from the sea hare Dolabella auricuralia, the shell-less opisthobranch mollusk. However, the dolabellanins are not small heat-labile proteins. Therefore, we performed experiments designed to find the antimicrobial peptides from D. auricuralia. An antifungal activity, that killed Saccharomyces cerevisiae after two hours coincubation, was extracted by 60% acetonitrile- 1% triofluoroacetate/water from skin and body wall. Further purification involved reverse-phase and cation exchange
OF GIANT
Koji Nakayama, Ann M. Nomoto, Miyuki Nishijima and Tadashi Maruyama.
Marine
Biotechnology
Laboratories,
B2. PURIFICATION OF ANTIFUNGAL ACTIVITY SEA HARE BODY WALL.
AND
Shimizu
Institute, 424,
Shimizu
Japan.
To
understand the cellular immunodefense system in the giant clam, Trdacna crocea, which harbors symbiotic microalga zooxanthella, hemocytes were characterized using light and electron microscopy. We observed of hemocytes: three types eosinophilic granulocytes, agranular cells and morula-like cells. Eosinophilic granulocytes contained granules approximately 0.6 pm in diameter. Agranular cells adhered to glass surfaces and contained electron lucent granules and few electron dense granules. Morula-like cells were packed with large (approximately 3 pm in diameter), electron dense granules. Eosinophilic granulocytes were acid phosphatase positive and were phagocytic against latex particles. Agranular cells and morula-like cells lacked phagocytic ability. When the hemolymph was exposed to seawater, hemocytes aggregated to form large clots and agranular cells were the main component found in the core of the clots. Morula-like cells released large granules during aggregation and they contained protein molecules having a molecular weight of 7.4 kDa. (Supported in a part by The Industrial Science and Technology Frontier Program by the New Energy and Industrial Technology Development Organization (Tokyo, Japan)). B4.
ALLO-RECOGNITION
AND
REJECTION OF SKIN GRAFTS IN THE TERRESTRIAL SLUG,
68
Zncilaria fruhstorferi. Yamaguchi, Emiko Hiroaki Nakamura*.
Eighth JADCI abstracts
Keiichiro Furuta* and i%e Laboratory of
fiuhstorferi
is composed chiefly of three cell types: 1) microvillous epithelial cells, 2) mucous cells, and 3) U-shaped cells. These Medical Sciences and *Department of are connected at their apical margins by Anatomy, Dokkyo University School of junctional complexes. In our ultrastructural Medicine, Mibu, Tochigi 321-02, Japan. In studies, we often observed that the U-shaped molluscs, the presence of allo-recognition has cells contained homogeneous components. not yet been proved, although heterotopic When carbon particles were injected into the transplantations have been tried. In the slug’s hemocoel, the particles passed rapidly present study, we transplanted auto- and through the skin. However, they were neither allografts of the dorsal skin to the same observed in the microvillous nor mucous Incilaria cells. We searched the route of passage of position in the land slug, fruhstotferi. Allo-rejection was judged using materials, using carbon particles, yeasts, a dissecting microscope and was characterized FITC-labeled dextran (MW 70 kDa), by disappearance of the graft outline and its hemocyanin antibodies and rhodamine-labeled shrinkage. In allografls, 76.9% of successful latex beads @=0.03p.m). Besides yeasts, grafts was rejected 12-26 days after these materials were found in tubule-like transplantation, while 62.5% of autografts spaces among the epithelial cells. Thirty min after injection of rhodamine-labeled latex survived for at least 26-44 days and statistical beads or FITC-labeled dextran, these indicated significant analysis (x2 -test) materials, which were contained in the body differences. By light microscopy, melanin surface mucus, were detected by fluorescence granules phagocytosed by macrophage-like cells were observed in allografis, but intact microscopy in the U-shaped cells. Seventy in melanocytes were observed in autografts. By hrs later, they were still distinguishable electron microscopy, a greater number of the mucus. These results revealed that the Uexogenous were found in shaped cells exudated these macrophage-like cells connective tissue of allografts when compared materials and endogenous hemocyanin (MW These materials might be to autografts and the number of mucous cells 400 kDa). markedly decreased in both alloand endocytosed and then released into the lumen autografts. Transplantation in molluscs is by exocytosis. difficult technically thus further observations and Host Defense in are essential in order to distinguish between (C) Hemocytes destroyed grafts caused by faulty technique vs Protochordates immuno-rejection. Cl. HISTOLOGICAL ANALYSIS HEMOCYTES AND OF B5. MOST SURPRISING CELLS TISSUE IN AMONG EPITHELIAL CELLS OF HEMOPOIETIC Halocynthia roretzi. Tomoo Sawada, WALL FROM THE BODY Nobuko Tokuda, Yoshihisa Fujikura Zncilaria TERRESTRIAL SLUG, and Tetsuo Fukumoto. Department of Furuta, Emiko fruhstorferi. Anatomy, Yamaguchi University School of Hiroaki Keiichiro Yamaguchi*, Medicine, Ube 755, Japan. We examined the Nakamura and Shin-ichi Kikuchi**. Department of Anatomy and *L.aboratory of distribution of various hemocytes in tissues of HaZocynthia roretzi, in order to improve Medical Science, Dokkyo University School in light-microscopic of Medicine, Mibu, Tochigi 321-02, Japan hemocyte identification and **Marine Ecosystems Research Center, tissue sections. Vacvolated cells remain inside the sinus-like spaces within connective Chiba University, Kominato, Chiba 299-55, Japan. Body wall epithelium of Incilaria tissue of the endocarp, but rarely infiltrated