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Seasonal changes in the multiple lectin compositions of the acorn barnacle Megabalanus rosa as related to ovarian developmetn
II
noted in granules of granulocytes and spberules of spherulocytes, respectively. No significant labeling was detectable on sections of probemocytes, piasmatocytes and oenocytoids. 3. SWITCHING OF HOST DEFENSE MECHANISMS IN RELATION TO METAMORPHOSIS OF INSECTS. Haruhisa Wage.
Delummem of Medical Technology, Saitama Medical School Junior College~Moroyama, Iruma-gun, Saitama 350-04, Japan. In Bomb~
Abstracts
after partial purification. Among the tissues tested, hemocytes were the major tissue for producing bemolymph iectins while ovary was also productive. Hemagglutinating activity was not detected in the culture medium of testis and fat body. We have previously indicated that an increase in iectin content in hemolymph seems to follow an increase in secretory activity of prothoracic gland. Control of its production by hemoc-ytes was e x a ~ e d using 4th instar larvae. The production of the lectin was enhanced by adding 2 0 - h y d ~ n e into the culture medium in a dose-dependent manner.
mot/, co-operation of both cellular and humoral defense factorsisessentialto eliminate invading organisms. The appearance and relative importance of those responding elements change dra- 5. ISOLATION OF eDNA CLONES CODING matically during ontogeny. This study examined FOR BOMBI~ LECTIN. Shin.iehl lwamoto, whether these aspects were applicable to wild Eqi Kotani, FuJlyoshi Matsubara, Motoyuki lepidopteran species Bomb~ mandar/na and Sumida and Hajime MorL Depam~nt of Pieris mpae crucivora. Results showed that :(1) Applied BIOlo~, Kyoto Institute of Technology, in both species the total number of hemocytes in- Kyoto 606, Jalum. In the hemolymph of the silkcreased during larval development and de- worm, Bomb~ mori, the concentration of hucreased to about one-half or one-third after meral lectin with bemagglutinating activity inpupation; (2) immunoeytessuch as granular cells creased at larval-larval ecdysis and spinning stage and plasmatoeytes increased their size at pupa- (Suzuki and Natori, J. Biochem. 93:583tion and the former lost their filopodia; (3) lectin 590;1983). It is induced by invasion of microoractivity in hemolymph appeared after pupation ganisrm and infection with cytoplasmic polyhewith concomitant expression of receptors for drosis virus (Mori et al., J. Invertebr. Pathol. 54: lectin only on granular cells. Furthermore, pre- 112-116;1989). It is highly probable that the injection of serine protease inhibitor into pupae Bomby~leedn served as a recognition molecule in in which lectin was present in hemolymph did not the defense system and participated in the ingesaffect phagocytic activity of bemoeytes against tion of decomposed tissues or cells by hemocytes. mammalian erythroeyteswhich were recognized A subunit with molecular weight of about by the lectin. Ligation experiments showed that 280kDa of the Bomb~ lectin was responsible for those drastic changes were under direct endo- hemagglutinating activity, since antiserum raised crine control. These results suggest that lepidop- against this polypeptide inhibited lectin activity. teran species adopt a switching strategy in host The site of synthesis of the BombFc lectin was defense to compensate for increase in cell size determined by primary tissue cultures of fat body with decreased numbers ofimmunocytes and the and bemocytes. Hemagglutinating activity assays new distribution of leetin receptor on granular demonstrated that hemocytes release a signifiphagocytes with the disappearance of membrane cant amount of hamagglutinin into the culture fitopodia. medium. Starting with poly(A)+RNA from spinning larval bemocytes, a eDNA library was pre4. THE SITE OF PRODUCTION OF pared in an expre~ion ve~or lamda gt11 and BOMBI'X HEMOLYMPH LECTIN AND several cDHA clones coding for the Bomb~ ACTIVATION OF ITS PRODUCTION BY lectin were isolated from the library. The eDNA 7~0-HYDROXYECDYSONE. Kazuhito clones were characterized. Amanai, She Sakurai* and Tetsuya Ohtaki*. NIBB, Okazaki 444, Japan and *Deparonem of 6. SEASONAL CHANGES IN THE MULBiology, Facultyof Science, Ka~_azawaUniversity, TIPLE LECTIN COMPOSITIONS OF THE Kan~.__z_a_wa920, Jatuut In order to identify the ACORN BARNACLE MEGABAI,dNUS ROSA source of leetin in larval hemolymph of Bomby~ AS RELATED TO OVARIAN DEVELOPmot/, ovary, testis, fat body and bemoeytes of 5th MENT. Koji Muramoto, Ryusuke Kado and instar larvae were cultured/n vitro and hemag- Hlaao Kamiya. School of Fisheries Sciences, glutinating activity in the medium was a:~yed I~asato Univers~y,S a w i ~ lwate 022.01, Japan.
Abstracts
Various invertebrate lectins within an individual and in different species may have diverse functions, as different as the structural features of the lectins themselves. Short.term variations oflectin levels after challenging invertebrates by injury or introducing microorganisms have been well studled, demonstrating the involvement of iectins in invertebrate immunity. On the other hand, little is known about the long-term variations of lectin levels. We found seasonal changes in multiple lectin compositions in coelomic fluid of the acorn barnacle Megabalanus rosa. The lectins were composed of three different molecular species with respect to molecular weight, designated BRA-1 (330kDa), BRA-2 (140kDa) and BRA-3 (64kDa). The total amount of lectins varied from 14 mg/100ml to 52 mg/100mi and exhibited ~ u m levels during early summer and M . Ovaries matured after this period. Annual cycles of ovarian development were similar to those of lectin levels, but the maxima were delayed several months after those of lectin levels. Immunocytochemical staining of the decalcified barnacle with speciflcM, rosa lectin antisera showed ~ossreaction in most tissues, particularly in cirri and connective tissues. 7. BIOCHEMICAL STUDIES ON HAEMOLYRIC AND PROTEOLYTIC FACTORS PRESENT IN THE BODY FLUID OF REDWORM, LUMBRICUS RUBF.LLUS. YongJong Son, Jeong-Woo Yi* and Chung-Soon Chang*. Department of Biology, College of
Science and * D e ~ n t of Biochemistry, Collegeof Medicine, Inha University,Inchon 402-751, Korea~ The body (or coelomic) fluid of the redworm, L. mbe/~s has strong hemolytic and proteolytic activities. According to our data, the novel hemolytic factor in this fluid originates from body tissue and is endowed with strong hemolysis against several mammalian erythrocytes,showing an average hemolytic titerof 3.3 x 103foldthe dilutionfactorby HA (hemagglutination) test.This factor also showed strong proteolytic activityagainst such proteins as human IgG, bovine serum albumin (BSA) and casein. To determine the biochemical characters of the hemolytic and proteolyfic factors, various physicochemical parameters were identified and we also searched for complement, protease and phospholipase inhibitors which commonly act on both factors. A symbiotic bacterium was isolated and cultured in a medium. It induced the production of yet another type of hemolytic factor differ-
III
ent from the one taken directly from the redworm's body fluid. 8. CHARACTERIZ&TION OF D-GALACTOSE-SPECI~C AGGLUTININ OF THE SOFT CORAL ,.q/NULAR/A SP. Rlna Goto, Koji Muramoto, Masatoshi Yamazaki*, and
Hlsao Kamly~ School of Fisheries Sciences, ICJtasato Universe, Sanriku-cho, Kesen-gu~ /.,ate 022-01,Japan and *Faculty of Phannaceutical Sciences, Teilg,ou Univeraity,Sagamiko-cho, Tsukui.gu~ Kanagawa 199.01, ]apa~ The aqueous extract of the soft coral Sinu/ar/a sp. collected in the vicinity of Fiji showed a hemagglutihating activity against rabbit erythrocytes but not against sheep or human A B e erythrocytes. A D-galactose-specLfic agglutinin was isolated by affinity chromatography on acid-treated Sepharose 4]3 and bygel filtration on a TSK G-3000SW column. The major component, designated sinularian, was a glycoprotein confining 11% sugar. It gave a single band corresponding to 78kDa in SDS-polyacrylamide gel electrophoresis, irrespective of treating it with 2-mercaptoethanol. Sinularian agglutinated rabbit erythrocytes and also routine leukemia cells, L1210 and L5178Y. Hemagglutinating activity was Ca**-independent. Hemagglutinating activity of sinularian against trypsinized rabbit erythrocytes was inhibRed by D-galactose, N-acetyl-D-galactosamine, and lactose. Sinularian showed no mitogenic activity but it promoted the binding of macrophages to tumor cells and inhibited cleavage of the sea urchin Hemicentrotus pulchetrimus at the blastula stage. 9. OPSONIZING EFFECT OF BODY SURFACE MUCUS IN THE LAND SLUG, INC3IARIA FURUHSTORFERI. Kelichlro Yamaguchi, ~miko Furuta* and Atsumi Shimozawa*. The Laboratory of Medical Sci-
ences and *Departmem of Anatomy, Dokkyo Unit~rsity School of Medicine, Mibt~ Tochigi 321.02, Japan. Macrophaga-like cells in hemolymph of the land slug phagocytose SRBC and latex beads in vivo and/n ~ , o . However, when these cells start to phagncyto~ foreign particles after injection through the body surface and whether or not the body surface mucus has an opeoniTJng effect on the cells have not been reported. In this study slugs were injected with SRBC treated with a muein fraction prepared from the body surface mucus. The ultrastructure of macrophage-like cells in slug hemolymph
noted in granules of granulocytes and spberules of spherulocytes, respectively. No significant labeling was detectable on sections of probemocytes, piasmatocytes and oenocytoids. 3. SWITCHING OF HOST DEFENSE MECHANISMS IN RELATION TO METAMORPHOSIS OF INSECTS. Haruhisa Wage.
Delummem of Medical Technology, Saitama Medical School Junior College~Moroyama, Iruma-gun, Saitama 350-04, Japan. In Bomb~
Abstracts
after partial purification. Among the tissues tested, hemocytes were the major tissue for producing bemolymph iectins while ovary was also productive. Hemagglutinating activity was not detected in the culture medium of testis and fat body. We have previously indicated that an increase in iectin content in hemolymph seems to follow an increase in secretory activity of prothoracic gland. Control of its production by hemoc-ytes was e x a ~ e d using 4th instar larvae. The production of the lectin was enhanced by adding 2 0 - h y d ~ n e into the culture medium in a dose-dependent manner.
mot/, co-operation of both cellular and humoral defense factorsisessentialto eliminate invading organisms. The appearance and relative importance of those responding elements change dra- 5. ISOLATION OF eDNA CLONES CODING matically during ontogeny. This study examined FOR BOMBI~ LECTIN. Shin.iehl lwamoto, whether these aspects were applicable to wild Eqi Kotani, FuJlyoshi Matsubara, Motoyuki lepidopteran species Bomb~ mandar/na and Sumida and Hajime MorL Depam~nt of Pieris mpae crucivora. Results showed that :(1) Applied BIOlo~, Kyoto Institute of Technology, in both species the total number of hemocytes in- Kyoto 606, Jalum. In the hemolymph of the silkcreased during larval development and de- worm, Bomb~ mori, the concentration of hucreased to about one-half or one-third after meral lectin with bemagglutinating activity inpupation; (2) immunoeytessuch as granular cells creased at larval-larval ecdysis and spinning stage and plasmatoeytes increased their size at pupa- (Suzuki and Natori, J. Biochem. 93:583tion and the former lost their filopodia; (3) lectin 590;1983). It is induced by invasion of microoractivity in hemolymph appeared after pupation ganisrm and infection with cytoplasmic polyhewith concomitant expression of receptors for drosis virus (Mori et al., J. Invertebr. Pathol. 54: lectin only on granular cells. Furthermore, pre- 112-116;1989). It is highly probable that the injection of serine protease inhibitor into pupae Bomby~leedn served as a recognition molecule in in which lectin was present in hemolymph did not the defense system and participated in the ingesaffect phagocytic activity of bemoeytes against tion of decomposed tissues or cells by hemocytes. mammalian erythroeyteswhich were recognized A subunit with molecular weight of about by the lectin. Ligation experiments showed that 280kDa of the Bomb~ lectin was responsible for those drastic changes were under direct endo- hemagglutinating activity, since antiserum raised crine control. These results suggest that lepidop- against this polypeptide inhibited lectin activity. teran species adopt a switching strategy in host The site of synthesis of the BombFc lectin was defense to compensate for increase in cell size determined by primary tissue cultures of fat body with decreased numbers ofimmunocytes and the and bemocytes. Hemagglutinating activity assays new distribution of leetin receptor on granular demonstrated that hemocytes release a signifiphagocytes with the disappearance of membrane cant amount of hamagglutinin into the culture fitopodia. medium. Starting with poly(A)+RNA from spinning larval bemocytes, a eDNA library was pre4. THE SITE OF PRODUCTION OF pared in an expre~ion ve~or lamda gt11 and BOMBI'X HEMOLYMPH LECTIN AND several cDHA clones coding for the Bomb~ ACTIVATION OF ITS PRODUCTION BY lectin were isolated from the library. The eDNA 7~0-HYDROXYECDYSONE. Kazuhito clones were characterized. Amanai, She Sakurai* and Tetsuya Ohtaki*. NIBB, Okazaki 444, Japan and *Deparonem of 6. SEASONAL CHANGES IN THE MULBiology, Facultyof Science, Ka~_azawaUniversity, TIPLE LECTIN COMPOSITIONS OF THE Kan~.__z_a_wa920, Jatuut In order to identify the ACORN BARNACLE MEGABAI,dNUS ROSA source of leetin in larval hemolymph of Bomby~ AS RELATED TO OVARIAN DEVELOPmot/, ovary, testis, fat body and bemoeytes of 5th MENT. Koji Muramoto, Ryusuke Kado and instar larvae were cultured/n vitro and hemag- Hlaao Kamiya. School of Fisheries Sciences, glutinating activity in the medium was a:~yed I~asato Univers~y,S a w i ~ lwate 022.01, Japan.
Abstracts
Various invertebrate lectins within an individual and in different species may have diverse functions, as different as the structural features of the lectins themselves. Short.term variations oflectin levels after challenging invertebrates by injury or introducing microorganisms have been well studled, demonstrating the involvement of iectins in invertebrate immunity. On the other hand, little is known about the long-term variations of lectin levels. We found seasonal changes in multiple lectin compositions in coelomic fluid of the acorn barnacle Megabalanus rosa. The lectins were composed of three different molecular species with respect to molecular weight, designated BRA-1 (330kDa), BRA-2 (140kDa) and BRA-3 (64kDa). The total amount of lectins varied from 14 mg/100ml to 52 mg/100mi and exhibited ~ u m levels during early summer and M . Ovaries matured after this period. Annual cycles of ovarian development were similar to those of lectin levels, but the maxima were delayed several months after those of lectin levels. Immunocytochemical staining of the decalcified barnacle with speciflcM, rosa lectin antisera showed ~ossreaction in most tissues, particularly in cirri and connective tissues. 7. BIOCHEMICAL STUDIES ON HAEMOLYRIC AND PROTEOLYTIC FACTORS PRESENT IN THE BODY FLUID OF REDWORM, LUMBRICUS RUBF.LLUS. YongJong Son, Jeong-Woo Yi* and Chung-Soon Chang*. Department of Biology, College of
Science and * D e ~ n t of Biochemistry, Collegeof Medicine, Inha University,Inchon 402-751, Korea~ The body (or coelomic) fluid of the redworm, L. mbe/~s has strong hemolytic and proteolytic activities. According to our data, the novel hemolytic factor in this fluid originates from body tissue and is endowed with strong hemolysis against several mammalian erythrocytes,showing an average hemolytic titerof 3.3 x 103foldthe dilutionfactorby HA (hemagglutination) test.This factor also showed strong proteolytic activityagainst such proteins as human IgG, bovine serum albumin (BSA) and casein. To determine the biochemical characters of the hemolytic and proteolyfic factors, various physicochemical parameters were identified and we also searched for complement, protease and phospholipase inhibitors which commonly act on both factors. A symbiotic bacterium was isolated and cultured in a medium. It induced the production of yet another type of hemolytic factor differ-
III
ent from the one taken directly from the redworm's body fluid. 8. CHARACTERIZ&TION OF D-GALACTOSE-SPECI~C AGGLUTININ OF THE SOFT CORAL ,.q/NULAR/A SP. Rlna Goto, Koji Muramoto, Masatoshi Yamazaki*, and
Hlsao Kamly~ School of Fisheries Sciences, ICJtasato Universe, Sanriku-cho, Kesen-gu~ /.,ate 022-01,Japan and *Faculty of Phannaceutical Sciences, Teilg,ou Univeraity,Sagamiko-cho, Tsukui.gu~ Kanagawa 199.01, ]apa~ The aqueous extract of the soft coral Sinu/ar/a sp. collected in the vicinity of Fiji showed a hemagglutihating activity against rabbit erythrocytes but not against sheep or human A B e erythrocytes. A D-galactose-specLfic agglutinin was isolated by affinity chromatography on acid-treated Sepharose 4]3 and bygel filtration on a TSK G-3000SW column. The major component, designated sinularian, was a glycoprotein confining 11% sugar. It gave a single band corresponding to 78kDa in SDS-polyacrylamide gel electrophoresis, irrespective of treating it with 2-mercaptoethanol. Sinularian agglutinated rabbit erythrocytes and also routine leukemia cells, L1210 and L5178Y. Hemagglutinating activity was Ca**-independent. Hemagglutinating activity of sinularian against trypsinized rabbit erythrocytes was inhibRed by D-galactose, N-acetyl-D-galactosamine, and lactose. Sinularian showed no mitogenic activity but it promoted the binding of macrophages to tumor cells and inhibited cleavage of the sea urchin Hemicentrotus pulchetrimus at the blastula stage. 9. OPSONIZING EFFECT OF BODY SURFACE MUCUS IN THE LAND SLUG, INC3IARIA FURUHSTORFERI. Kelichlro Yamaguchi, ~miko Furuta* and Atsumi Shimozawa*. The Laboratory of Medical Sci-
ences and *Departmem of Anatomy, Dokkyo Unit~rsity School of Medicine, Mibt~ Tochigi 321.02, Japan. Macrophaga-like cells in hemolymph of the land slug phagocytose SRBC and latex beads in vivo and/n ~ , o . However, when these cells start to phagncyto~ foreign particles after injection through the body surface and whether or not the body surface mucus has an opeoniTJng effect on the cells have not been reported. In this study slugs were injected with SRBC treated with a muein fraction prepared from the body surface mucus. The ultrastructure of macrophage-like cells in slug hemolymph