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G2.P3 Prophenoloxidase-activating system strongly affects the initial defensive function of Bombyx mori granulocytes
The Scientific and Social Program of the VFh ISDCI Congress
$1 2 9
G2.P3 PROPHENOLOXIDASE-ACTIVATING SYSTEM STRONGLY AFFECTS INITIAL DEFENSIVE FUNCTION OF BOMBYX MORI GRANULOCYTES
THE
Haruhisa Wago Laboratory of Immunology, Department of Medical Technology, Saitama Medical School Junior College, Moroyana, Iruma-gun, Saitama 35004, Japan Lectin or prophenoloxidase-activating system (proPO-AS) is considered to be involved in the cellular response by insect granulocytes to eliminate the invading foreign materials. This study was carried out to examine to which extent the proPO-AS participates in the initial cellular defense reaction using oxidase or serine protease inhibitors and anticoagulant. In the presence of serine protease inhibitor, only plasmatocytes could attach to the foreign surface. However, anticoagulant completely inhibited the attachment of both granulocytes and plasmatocytes. Additionally, the treatment of foreign surface with melanized hemolymph greatly promoted the attachment of granulocyte. This foreign surface showed strong formation of dopachrome, suggesting that certain factor in proPO-AS adhered to foreign surface as a cell-adhesion molecule. This adhesion-promoting factor was extracted and SDS-PAGE analysis showed three kinds of proteins, about 31 kDa, 38 kDa and 7 0 kDa. A 70 kDa protein is known to be a phenoloxidase in B. mori, indicating involvement of this protein in cell-adhesion by granulocytes. Phagocytosis of yeast particles treated with kitalase, a yeast cell-lytic /~-l,3-glucanase, by granulocytes was remarkably decreased. Thus, it is concluded that proPO-AS strongly affects the initial attachment phase of granulocytes to foreign materials in B. mori.
G2.P4 A FOUR-DOMAIN
KAZAL
INHIBITOR
FROM
CRAYFISH
BLOOD
CELLS
Pia Keyser, Mats W. Johansson, Kenneth S6derh~ll Department of Physiological Botany, University of Uppsala, Villav/igen 6, S-752 27 Uppsala, Sweden A four domain Kazal inhibitor-like sequence was found in a cDNA library constructed from blood cells of the crayfish Pacifastacus leniusculus. It has an open reading frame of 684 base pairs and a signal sequence. The mature protein has a calculated mass of 22.7 kDa. Within the amino acid sequence there are four repeated streches, 45-73% identical to each other. These domains have high similarity to serine proteinase inhibitors of the Kazal family. The higest similarity was found between the first domain in the crayfish inhibitor and the one-domain elastase inhibitor from the sea anemone Anemonia sulcate. The first three domains have a leucine residue in the putative site, suggesting that the protein is a chymotrypsin inhibitor. From blood cells of crayfish a 23 kDa protein was found to bind to a chymotrypsin affinity column when blood cell homogenate was added. By amino terminal sequencing it was confirmed to be the cloned Kazal inhibitor. The purified protein inhibited the activity of the serine proteinase chymotrypsin at 1: 1 molar ratio. It also affected the activity of subtilisin but to a lesser extent and had no effect on elsastase, trypsin or thrombin.
$1 2 9
G2.P3 PROPHENOLOXIDASE-ACTIVATING SYSTEM STRONGLY AFFECTS INITIAL DEFENSIVE FUNCTION OF BOMBYX MORI GRANULOCYTES
THE
Haruhisa Wago Laboratory of Immunology, Department of Medical Technology, Saitama Medical School Junior College, Moroyana, Iruma-gun, Saitama 35004, Japan Lectin or prophenoloxidase-activating system (proPO-AS) is considered to be involved in the cellular response by insect granulocytes to eliminate the invading foreign materials. This study was carried out to examine to which extent the proPO-AS participates in the initial cellular defense reaction using oxidase or serine protease inhibitors and anticoagulant. In the presence of serine protease inhibitor, only plasmatocytes could attach to the foreign surface. However, anticoagulant completely inhibited the attachment of both granulocytes and plasmatocytes. Additionally, the treatment of foreign surface with melanized hemolymph greatly promoted the attachment of granulocyte. This foreign surface showed strong formation of dopachrome, suggesting that certain factor in proPO-AS adhered to foreign surface as a cell-adhesion molecule. This adhesion-promoting factor was extracted and SDS-PAGE analysis showed three kinds of proteins, about 31 kDa, 38 kDa and 7 0 kDa. A 70 kDa protein is known to be a phenoloxidase in B. mori, indicating involvement of this protein in cell-adhesion by granulocytes. Phagocytosis of yeast particles treated with kitalase, a yeast cell-lytic /~-l,3-glucanase, by granulocytes was remarkably decreased. Thus, it is concluded that proPO-AS strongly affects the initial attachment phase of granulocytes to foreign materials in B. mori.
G2.P4 A FOUR-DOMAIN
KAZAL
INHIBITOR
FROM
CRAYFISH
BLOOD
CELLS
Pia Keyser, Mats W. Johansson, Kenneth S6derh~ll Department of Physiological Botany, University of Uppsala, Villav/igen 6, S-752 27 Uppsala, Sweden A four domain Kazal inhibitor-like sequence was found in a cDNA library constructed from blood cells of the crayfish Pacifastacus leniusculus. It has an open reading frame of 684 base pairs and a signal sequence. The mature protein has a calculated mass of 22.7 kDa. Within the amino acid sequence there are four repeated streches, 45-73% identical to each other. These domains have high similarity to serine proteinase inhibitors of the Kazal family. The higest similarity was found between the first domain in the crayfish inhibitor and the one-domain elastase inhibitor from the sea anemone Anemonia sulcate. The first three domains have a leucine residue in the putative site, suggesting that the protein is a chymotrypsin inhibitor. From blood cells of crayfish a 23 kDa protein was found to bind to a chymotrypsin affinity column when blood cell homogenate was added. By amino terminal sequencing it was confirmed to be the cloned Kazal inhibitor. The purified protein inhibited the activity of the serine proteinase chymotrypsin at 1: 1 molar ratio. It also affected the activity of subtilisin but to a lesser extent and had no effect on elsastase, trypsin or thrombin.