- Email: [email protected]
12. Purification of cytolytic proteins from purple and body fluid of the sea hare, Dolabella auricularia
IV
collected at 1, 3, 6 and 24 hr after the injection was observed by transmission electron microscopy. The macrophage-llke cells phagocytosed several SRBC treated with the mucin fraction at 1, 3 and 6 hr after injection. When non-treated SRBC were injected, however, few hemocytes phagocytosed at 3 and 6 hr after injection. These results suggest that the body surface mucusofthe land slug contains opsonizing factors which enhance the phagocytosis of foreign particles by macrophage-like cells in the hemolymph.
Abstracts
hemocyte capsules around the parasite were studied in M-iineBiomphalariaglabrata snails exposed to 100 or 400 first-stage larvae of Anglos. trongylus cantonensis. Snails exposed to 100 iar. vae showed significant increases in circulating hemocytes at 1 day post-exposure (dpe). The number of hemocytes remained significantly elevated through 14 dpe, declined to control values by 35 dpe, and increased again at 42 dpe. In snails exposed to 400 larvae, a significant increase in number of hemocytes was also noted at 1 dpe. The number of hemocytes was lower than in 10. LECTIN-LIKE ACTIVITY OF THE unexposed snails at 7 dpe, then increased conBODY SURFACE MUCUS AND THE stantly by 42 dpe. The decrease in number of HEMOLYMPH OF THE LAND SLUG, IN. circulating bemocytes is probably due to the CILARIA FRUHSTORFERI. Emiko Furuta, removal of cells from the circulation to particiKeiichiro Yamaguchi* and Atsumi pate in encapsulation of larvae. The majority of Shimozawa. DeparlmentofAnatomy and *the circulating hemocytes in M-line B. glabrata are Laboratory of Medical Sciences, Doldcyo Univer- fully-spread granulocytes, which increase signifis ~ School of Medicine, Mib~ Tochigi 321-02, cantly in number in snails following exposure to Japa~ How slugs protect their bodies from in- A.cantonensis larvae. However, a population of fection by microorganisms is an interesting prob- partially-spread granulocytes, round cells, hyalilem. In spite of their moist and fragile cutaneous nocytes and miscellaneous hemocytes were relatissues, slugs seem to be protected from most tively constant. The size of capsules around the bacterial infections. Body surface mucus was col- parasite increased during the 42-days interval of lected by gently prodding a relaxed body with a the experiment. The hematopoietic organ inblunt glass rod. The mucus was diluted with 5 vol creased in size in response to infection. of water, stirred overnight and centrifuged at 10,000xg for 2 hr at 4°(2 to remove insoluble 12. PURIFICATION OF CYTOLYTIC PROmaterials (WSF). Hemolymph was collected TEINS FROM PURPLE AND BODY FLUID from the mantle cavity using a syringe with a 27- OF THE SEA HARE, DOLABELLA AURICUgauge needle (SH). WSF agglutinated human A LAR/A. Masatoshi Yamazaki, Jun Kisugi and and B type erythrocytes, and WSF induced I-Iisao Kamiya*. Facultyof Pharmaceutical Scihemagglutination was, however, specifically in- ences, Teikyo University, Sagamiko-cho, Ka. hibited by GalNac (70 pM). WSF also caused nagawa 199-01, Japan and *Laboratory of Mahemolysis of B type HRBC 12 hr after agglutina- rine Biochemical Resources, School of Fisheries tion. All these WSF activities were completely Sciences, IOtasato University, Sanriku, Iwate 022inactivated by heating at 56°C for 30 rain. SH 01, Japan. Acytolytic factor, dolabeilanin P, was agglutinated A and B type HRBC, and GalNac purified to apparent homogeneity from the and NeuNac effectively inhibited the hemaggiuti- purple fluid of the sea hare, Do/abe//a auricunation. These results suggest that the body sur- /ar/a. Purified dolabeUanin P was a single poiface mucus and hemolymph of land slugs con- ypeptide of 60 kDa. This factor nonspecifically tained lectin-Uke activity. lysed routine MM46, MH134 and meth A tumor cells within 2 hr. Another antineoplastic factor, 11. EFFECTS OF INFECTION WITH ANGI. dolabeUanin C, inducing tumor lysis was also OSTRONGYLUS CANTONENSIS ON THE purified to apparent homogeneity. Purified dolaCIRCULATING HAEMOCYTE POPULA- bellanin C was a glycoprotein of 215 kDa consistTION AND THE HAEMATOPOIETIC ingof3subunitsof70 kDa.Thls factorwasactive ORGAN OF THE HOST SNAIL M-LINE against tumor cells even at 0.38 ng protein/ml and BIOMPflALa4RIA GLABRATA. Shiniehi Noda. caused complete cytolysis within 18 hr. These Deparonent of Medical Zoology, Facultyof Medi- factors (dolabellanin P and C) are distinct from cinc, Kagoshima University, Kagoshima 890, Ja- antineoplastic glycoproteins previously isolated pan. The number of circulating hemocytes, the from eggs (aplysianin E) or albumin glands (aplysize of the hematopoietic organ, and the size of slaninin A) ofAp/ys/a kuroda/ in terms of certain
Abstracts
V
cytolytic properties. These results suggest that dolabellanin P and C, found in the sea hare, a marine invertebrate, are new cytolytic factors.
umn, the ink of a cuttlefish was fractionated into two fractions that were eluted with 50 mM NaCI solution (peak 1) and 360 mM NaCI solution (peak 2). Next, these factors were purified by 13. SURVIVAL OF VIBRIO PARAHAEMO- Sephacryi S-200. Peak 1 was separated into two LYTICUS AND ESCHERICHIA COLI IN peaks which were eluted at 240 kDa and 90 kDa. THREE NERITID MOLLUSCS. Norichika Peak 2 was eluted at 250 kDa. When these parHari Kumazawa, Ken lwao and Ellchi Kato. tially purified fractions were analysed on SDSDepartment of VeterinaryPublic Health, Faculty polyaerylamide gel electrophoresis, the factors of Agriculture, Tottori University, Tottori 680, showed 3 main bands of 86 kDa, 29 kDa and 53 Japan. VibHo parahaemolvt/cus is a bacterium kDa. These partially purified factorslysedM M 4 6 which can cause food poisoning in man by inges- tumor cellsat a low concentration (protein contion of seafoods. In our experiments, E para- centration 1 pg/ml). haemo/yt/cus has been detected at high levels from an estuarine neritid gastropod, Clithon 15. THE PHAGOCYTES IN HEMOLYMPH retrop/ctus. In the present study, the survival of OF HALOCYNTH/,'I RORETZI. Shin-ichi this bacterium was compared with C. retrop/ctus Ohtake, Takayuki Abe, Fumio Shishlkura and and two marine neritids, Heminerita japonica Kunio Tanaka. Department of Biology, Nihon and Nerita albicilla. Two strains (D-3 and R-13) UniversitySchool ofMedicinc, Tokyo 173,Japan. of V. parahaemo/yt/cus survived in C. retropicus Latex beads (1 p m ¢0injected into the hemocoel at a level of 104-105 viable units per gram for at were cleared from hemolymph plasma and found least 21 days. C. retrop/cus were maintained in ar- in hemocytes 2 hr after injection. Then, identifitificial seawater, adjusted the salinities to 15 and cation of phagocytic cells and possible targets 20 per rail at 25"C. On the other hand V. para- were studied/n v/tro. Latex beads(LB) (1, 5 and haemo/yt/cus were eliminated within 3 days from 25/~m ¢,), giumraldehyde-treated SRBC and E. N. albici//a maintained in 35 per rail artificial sea- cold (Ee) were used as target particles. The parwater and within 7 days from H. japonica main- ticles suspended in artificial sea water (HEPES, mined in 15-35 per rail artificial seawater.Escher- pH7.2) were incubated for a certain minute (1ichia coldYS-2 appeared to survive in C. retropic- 30 rain.) at 230C with an aliquot of freshly coltus and H. japonica at a level of 102-103 viable lected hemolymph. Phagocytic processes were units per gram for at least 14 days. As salinity examined by phase contrast and electron milevels ofhabitatswhieh consisted in C. retropietus, ercacopy. Small granular amebocytes (SO) acH. japonica and N. albicilla were 0-34, 10-41 and tively ingested all kinds of targets except LB-25 30-39 per rail water respectively, the defense /~m. A SG seemed to begin phagocytosis by its system of these molluscs against these bacteria pseudopod immediately after contact with LB, might be elucidated on the relationship to their Ec or SRBC and finished it within 30 sec. Eightpreferable salinities. een percent of SG ingested LB-1/~m within 1 rain and 88% of them did during 30 rain. Large LB 14. PURIFICATION OF ANTINEOPLASTIC (25/~m ¢,) were not ingested but encapsulated FACTOR FROM INK OF A CUTTLEFISH, with SG. Large granular amebocytes(LG) also TODARODES PACIFICUS. Jun Kisugi, showed phagocytosls though the percentage of Masatoshi Yamazaki and Hisao Kamlya*. LG ingesting particles during 30 rain of incubaMedical Chemistry, Faculty of Pharmaceutical tion was only 4-8% and targets were limited to Sciences, Teikyo University, Sagamiko, Ka- small particles such as LB-1/~m. We found no nagawa 199-01, Japan and *Laboratoryof Bio- particles in other hemocytes.
chemicalResources, School ofFisheriesSciences, IOtasato University,Sanrilcu,Iwate022-01,Japan. We found that the ink of a cuttlefish, Todarodes pacificus, has antineoplastic activity. The ink of a cuttlefish lysed tumor cells at a 1,000 dilution. Three antineoplastic factors were partially purified from the ink of a cuttlefish by ion-exchange chromatography and gel filtration. During the first step of purification on a DEAE-celluiose col-
16. CLASSIFICATION AND SOME CHAR. ACTERISTICS OF H E M O C Y T ~ OF THE ASCIDIA HALOCYNTHIA RORETZI. Tomoo Sawada, Yoshlhisa Fujikura, Susumu Tomonaga* and Tetsuo Fukumoto. Yama-
guchi UniversitySchool ofMedicine, Ube 755 and *School of Allied Health Sciences, Yamaguchi University, Ube 755, Japan. According to vital
collected at 1, 3, 6 and 24 hr after the injection was observed by transmission electron microscopy. The macrophage-llke cells phagocytosed several SRBC treated with the mucin fraction at 1, 3 and 6 hr after injection. When non-treated SRBC were injected, however, few hemocytes phagocytosed at 3 and 6 hr after injection. These results suggest that the body surface mucusofthe land slug contains opsonizing factors which enhance the phagocytosis of foreign particles by macrophage-like cells in the hemolymph.
Abstracts
hemocyte capsules around the parasite were studied in M-iineBiomphalariaglabrata snails exposed to 100 or 400 first-stage larvae of Anglos. trongylus cantonensis. Snails exposed to 100 iar. vae showed significant increases in circulating hemocytes at 1 day post-exposure (dpe). The number of hemocytes remained significantly elevated through 14 dpe, declined to control values by 35 dpe, and increased again at 42 dpe. In snails exposed to 400 larvae, a significant increase in number of hemocytes was also noted at 1 dpe. The number of hemocytes was lower than in 10. LECTIN-LIKE ACTIVITY OF THE unexposed snails at 7 dpe, then increased conBODY SURFACE MUCUS AND THE stantly by 42 dpe. The decrease in number of HEMOLYMPH OF THE LAND SLUG, IN. circulating bemocytes is probably due to the CILARIA FRUHSTORFERI. Emiko Furuta, removal of cells from the circulation to particiKeiichiro Yamaguchi* and Atsumi pate in encapsulation of larvae. The majority of Shimozawa. DeparlmentofAnatomy and *the circulating hemocytes in M-line B. glabrata are Laboratory of Medical Sciences, Doldcyo Univer- fully-spread granulocytes, which increase signifis ~ School of Medicine, Mib~ Tochigi 321-02, cantly in number in snails following exposure to Japa~ How slugs protect their bodies from in- A.cantonensis larvae. However, a population of fection by microorganisms is an interesting prob- partially-spread granulocytes, round cells, hyalilem. In spite of their moist and fragile cutaneous nocytes and miscellaneous hemocytes were relatissues, slugs seem to be protected from most tively constant. The size of capsules around the bacterial infections. Body surface mucus was col- parasite increased during the 42-days interval of lected by gently prodding a relaxed body with a the experiment. The hematopoietic organ inblunt glass rod. The mucus was diluted with 5 vol creased in size in response to infection. of water, stirred overnight and centrifuged at 10,000xg for 2 hr at 4°(2 to remove insoluble 12. PURIFICATION OF CYTOLYTIC PROmaterials (WSF). Hemolymph was collected TEINS FROM PURPLE AND BODY FLUID from the mantle cavity using a syringe with a 27- OF THE SEA HARE, DOLABELLA AURICUgauge needle (SH). WSF agglutinated human A LAR/A. Masatoshi Yamazaki, Jun Kisugi and and B type erythrocytes, and WSF induced I-Iisao Kamiya*. Facultyof Pharmaceutical Scihemagglutination was, however, specifically in- ences, Teikyo University, Sagamiko-cho, Ka. hibited by GalNac (70 pM). WSF also caused nagawa 199-01, Japan and *Laboratory of Mahemolysis of B type HRBC 12 hr after agglutina- rine Biochemical Resources, School of Fisheries tion. All these WSF activities were completely Sciences, IOtasato University, Sanriku, Iwate 022inactivated by heating at 56°C for 30 rain. SH 01, Japan. Acytolytic factor, dolabeilanin P, was agglutinated A and B type HRBC, and GalNac purified to apparent homogeneity from the and NeuNac effectively inhibited the hemaggiuti- purple fluid of the sea hare, Do/abe//a auricunation. These results suggest that the body sur- /ar/a. Purified dolabeUanin P was a single poiface mucus and hemolymph of land slugs con- ypeptide of 60 kDa. This factor nonspecifically tained lectin-Uke activity. lysed routine MM46, MH134 and meth A tumor cells within 2 hr. Another antineoplastic factor, 11. EFFECTS OF INFECTION WITH ANGI. dolabeUanin C, inducing tumor lysis was also OSTRONGYLUS CANTONENSIS ON THE purified to apparent homogeneity. Purified dolaCIRCULATING HAEMOCYTE POPULA- bellanin C was a glycoprotein of 215 kDa consistTION AND THE HAEMATOPOIETIC ingof3subunitsof70 kDa.Thls factorwasactive ORGAN OF THE HOST SNAIL M-LINE against tumor cells even at 0.38 ng protein/ml and BIOMPflALa4RIA GLABRATA. Shiniehi Noda. caused complete cytolysis within 18 hr. These Deparonent of Medical Zoology, Facultyof Medi- factors (dolabellanin P and C) are distinct from cinc, Kagoshima University, Kagoshima 890, Ja- antineoplastic glycoproteins previously isolated pan. The number of circulating hemocytes, the from eggs (aplysianin E) or albumin glands (aplysize of the hematopoietic organ, and the size of slaninin A) ofAp/ys/a kuroda/ in terms of certain
Abstracts
V
cytolytic properties. These results suggest that dolabellanin P and C, found in the sea hare, a marine invertebrate, are new cytolytic factors.
umn, the ink of a cuttlefish was fractionated into two fractions that were eluted with 50 mM NaCI solution (peak 1) and 360 mM NaCI solution (peak 2). Next, these factors were purified by 13. SURVIVAL OF VIBRIO PARAHAEMO- Sephacryi S-200. Peak 1 was separated into two LYTICUS AND ESCHERICHIA COLI IN peaks which were eluted at 240 kDa and 90 kDa. THREE NERITID MOLLUSCS. Norichika Peak 2 was eluted at 250 kDa. When these parHari Kumazawa, Ken lwao and Ellchi Kato. tially purified fractions were analysed on SDSDepartment of VeterinaryPublic Health, Faculty polyaerylamide gel electrophoresis, the factors of Agriculture, Tottori University, Tottori 680, showed 3 main bands of 86 kDa, 29 kDa and 53 Japan. VibHo parahaemolvt/cus is a bacterium kDa. These partially purified factorslysedM M 4 6 which can cause food poisoning in man by inges- tumor cellsat a low concentration (protein contion of seafoods. In our experiments, E para- centration 1 pg/ml). haemo/yt/cus has been detected at high levels from an estuarine neritid gastropod, Clithon 15. THE PHAGOCYTES IN HEMOLYMPH retrop/ctus. In the present study, the survival of OF HALOCYNTH/,'I RORETZI. Shin-ichi this bacterium was compared with C. retrop/ctus Ohtake, Takayuki Abe, Fumio Shishlkura and and two marine neritids, Heminerita japonica Kunio Tanaka. Department of Biology, Nihon and Nerita albicilla. Two strains (D-3 and R-13) UniversitySchool ofMedicinc, Tokyo 173,Japan. of V. parahaemo/yt/cus survived in C. retropicus Latex beads (1 p m ¢0injected into the hemocoel at a level of 104-105 viable units per gram for at were cleared from hemolymph plasma and found least 21 days. C. retrop/cus were maintained in ar- in hemocytes 2 hr after injection. Then, identifitificial seawater, adjusted the salinities to 15 and cation of phagocytic cells and possible targets 20 per rail at 25"C. On the other hand V. para- were studied/n v/tro. Latex beads(LB) (1, 5 and haemo/yt/cus were eliminated within 3 days from 25/~m ¢,), giumraldehyde-treated SRBC and E. N. albici//a maintained in 35 per rail artificial sea- cold (Ee) were used as target particles. The parwater and within 7 days from H. japonica main- ticles suspended in artificial sea water (HEPES, mined in 15-35 per rail artificial seawater.Escher- pH7.2) were incubated for a certain minute (1ichia coldYS-2 appeared to survive in C. retropic- 30 rain.) at 230C with an aliquot of freshly coltus and H. japonica at a level of 102-103 viable lected hemolymph. Phagocytic processes were units per gram for at least 14 days. As salinity examined by phase contrast and electron milevels ofhabitatswhieh consisted in C. retropietus, ercacopy. Small granular amebocytes (SO) acH. japonica and N. albicilla were 0-34, 10-41 and tively ingested all kinds of targets except LB-25 30-39 per rail water respectively, the defense /~m. A SG seemed to begin phagocytosis by its system of these molluscs against these bacteria pseudopod immediately after contact with LB, might be elucidated on the relationship to their Ec or SRBC and finished it within 30 sec. Eightpreferable salinities. een percent of SG ingested LB-1/~m within 1 rain and 88% of them did during 30 rain. Large LB 14. PURIFICATION OF ANTINEOPLASTIC (25/~m ¢,) were not ingested but encapsulated FACTOR FROM INK OF A CUTTLEFISH, with SG. Large granular amebocytes(LG) also TODARODES PACIFICUS. Jun Kisugi, showed phagocytosls though the percentage of Masatoshi Yamazaki and Hisao Kamlya*. LG ingesting particles during 30 rain of incubaMedical Chemistry, Faculty of Pharmaceutical tion was only 4-8% and targets were limited to Sciences, Teikyo University, Sagamiko, Ka- small particles such as LB-1/~m. We found no nagawa 199-01, Japan and *Laboratoryof Bio- particles in other hemocytes.
chemicalResources, School ofFisheriesSciences, IOtasato University,Sanrilcu,Iwate022-01,Japan. We found that the ink of a cuttlefish, Todarodes pacificus, has antineoplastic activity. The ink of a cuttlefish lysed tumor cells at a 1,000 dilution. Three antineoplastic factors were partially purified from the ink of a cuttlefish by ion-exchange chromatography and gel filtration. During the first step of purification on a DEAE-celluiose col-
16. CLASSIFICATION AND SOME CHAR. ACTERISTICS OF H E M O C Y T ~ OF THE ASCIDIA HALOCYNTHIA RORETZI. Tomoo Sawada, Yoshlhisa Fujikura, Susumu Tomonaga* and Tetsuo Fukumoto. Yama-
guchi UniversitySchool ofMedicine, Ube 755 and *School of Allied Health Sciences, Yamaguchi University, Ube 755, Japan. According to vital