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D.P5 Activation of the complement system of the marsupial Monodelphis domestica
The Scientific and Social Program of the V1~ ISDC! Congress
985
D.P5 ACTIVATION
OF
THE
COMPLEMENT
SYSTEM
OF
THE
MARSUPIAL
M O N O D E L P H I S DOMESTICA T.L. Koppenheffer, Heather M. Falvo, Kay D. Spong Dept. of Biology, Trinity University, San Antonio, TX 78212, USA We have begun investigating the complement system of the South American marsupial Monodelphis domestica by determining the requirements for activation of the classical and alternative complement pathways. Using sheep erythrocytes sensitized with rabbit hemolysin to study the classical pathway, and unsensitized rabbit erythrocytes to study the alternative pathway, we found the former to require both Ca and Mg ions whereas only Mg ions were required by the latter. These activation pathways displayed similar kinetics of hemolysis, but showed marked differences in other properties. Hemolytic activity induced by the classical pathway was maximal at an ionic strength of 0.083 and a temperature of 30 °, whereas the alternative activation pathway functioned optimally at lower ionic strengths and displayed a broad temperature optimum between 34 ° and 37 ° . The classical activation pathway was highly susceptible to heat inactivation, losing > 50 % of its activity after 40 rain. at 40 °, whereas the alternative pathway retained >90% of its activity under the same conditions. The lytic capacity of both complement pathways was reduced by prior incubation with zymosan and inulin, whereas only the classical activation pathway was inhibited by exposure to carageenan. Thus, the complement activation pathways of this marsupial exhibit divalent cation requirements and reactivity with complement activators which are similar to those of placental mammals. At the same time, there exist several characteristics of both activation pathways which may be unique to marsupials and may reflect physiological adaptations of this group of mammals.
D.P6 A HEMOCYTE PROTEINASE PARTICIPATING LARVAL FAT BODY DURING METAMORPHOSIS (FLESH FLY)
IN DECOMPOSITION OF THE O F SARCOPHAGA P E ~ G R I N A
Shoichiro Kurata, Shunji Natori Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan In holometabolous insects, most larval tissues are decomposed in the pupal stage. Nothing is known about the molecular mechanism of selective decomposition of the larval tissues. However, we recently demonstrated that, in Sarcophagaperegrina, hemocytes acquire the ability to dissociate the larval fat body during pupation. This is a surprising phenomenon from the immunological point of view, because it indicates that self cells change their character to attack self tissues in this ontogenetic process. In this report, we examined how pupal hemocytes dissociate the larval fat body. The following findings suggest that for dissociation of the fat body, hemocytes secrete a proteinase, which is an insect cathepsin B, when they interact with the fat body. (1) Dissociation of the fat body by pupal hemocytes was inhibited by chymostatin. Of the various proteinases so far tested, only chymotrypsin dissociated the fat body, mimicks the activity of pupal hemocytes. (2) Using a substrate for chymotrypsin, we purified a 29-kDa proteinase from pupal hemocytes. Antibody against this proteinase repressed the dissociation of the fat body by pupal hemocytes. (3) Fat body was dissociated when incubated with the purified enzyme in vitro. (4) This proteinase was localized in granules of heterogeneous size in the hemocytes and was released when they incubated with the fat body. (5) The amino acid sequence of this enzyme showed significant similarity to that of mammlian cathepsin B.
985
D.P5 ACTIVATION
OF
THE
COMPLEMENT
SYSTEM
OF
THE
MARSUPIAL
M O N O D E L P H I S DOMESTICA T.L. Koppenheffer, Heather M. Falvo, Kay D. Spong Dept. of Biology, Trinity University, San Antonio, TX 78212, USA We have begun investigating the complement system of the South American marsupial Monodelphis domestica by determining the requirements for activation of the classical and alternative complement pathways. Using sheep erythrocytes sensitized with rabbit hemolysin to study the classical pathway, and unsensitized rabbit erythrocytes to study the alternative pathway, we found the former to require both Ca and Mg ions whereas only Mg ions were required by the latter. These activation pathways displayed similar kinetics of hemolysis, but showed marked differences in other properties. Hemolytic activity induced by the classical pathway was maximal at an ionic strength of 0.083 and a temperature of 30 °, whereas the alternative activation pathway functioned optimally at lower ionic strengths and displayed a broad temperature optimum between 34 ° and 37 ° . The classical activation pathway was highly susceptible to heat inactivation, losing > 50 % of its activity after 40 rain. at 40 °, whereas the alternative pathway retained >90% of its activity under the same conditions. The lytic capacity of both complement pathways was reduced by prior incubation with zymosan and inulin, whereas only the classical activation pathway was inhibited by exposure to carageenan. Thus, the complement activation pathways of this marsupial exhibit divalent cation requirements and reactivity with complement activators which are similar to those of placental mammals. At the same time, there exist several characteristics of both activation pathways which may be unique to marsupials and may reflect physiological adaptations of this group of mammals.
D.P6 A HEMOCYTE PROTEINASE PARTICIPATING LARVAL FAT BODY DURING METAMORPHOSIS (FLESH FLY)
IN DECOMPOSITION OF THE O F SARCOPHAGA P E ~ G R I N A
Shoichiro Kurata, Shunji Natori Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan In holometabolous insects, most larval tissues are decomposed in the pupal stage. Nothing is known about the molecular mechanism of selective decomposition of the larval tissues. However, we recently demonstrated that, in Sarcophagaperegrina, hemocytes acquire the ability to dissociate the larval fat body during pupation. This is a surprising phenomenon from the immunological point of view, because it indicates that self cells change their character to attack self tissues in this ontogenetic process. In this report, we examined how pupal hemocytes dissociate the larval fat body. The following findings suggest that for dissociation of the fat body, hemocytes secrete a proteinase, which is an insect cathepsin B, when they interact with the fat body. (1) Dissociation of the fat body by pupal hemocytes was inhibited by chymostatin. Of the various proteinases so far tested, only chymotrypsin dissociated the fat body, mimicks the activity of pupal hemocytes. (2) Using a substrate for chymotrypsin, we purified a 29-kDa proteinase from pupal hemocytes. Antibody against this proteinase repressed the dissociation of the fat body by pupal hemocytes. (3) Fat body was dissociated when incubated with the purified enzyme in vitro. (4) This proteinase was localized in granules of heterogeneous size in the hemocytes and was released when they incubated with the fat body. (5) The amino acid sequence of this enzyme showed significant similarity to that of mammlian cathepsin B.