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D.P11 Uptake of gold-labeled lectins (wga, con a) by leucocytes of the carp (Cyprinus carpio L.)
988
D.Pll UPTAKE
The Scientific and Social Program of the W h ISDCI Congress
OF GOLD-LABELED
LECTINS
( W G A , C O N A) B Y L E U C O C Y T E S
OF
T H E C A R P (CYPRINUS CARPIO L.) Bielek Edith Histological & Embryological Institute, University of Vienna, Schwarzspanierstr. 17, A-1090 Vienna, Austria In lymphocytes, the internalization of macromolecules and membrane components plays an important role in activation and regulation. Lectins binding to the surface glycoproteins and mediating different functions may be used as markers. The uptake of WGA (wheat germ agglutinin) by human cytotoxic lymphocytes was observed after light microscopic fluorescence labeling (1), as well as the preferential binding of WGA by natural killer cells (2). In the present study, binding and uptake of gold-labeled lectins were studied electron-microscopically in the carp. Leucocytes of peripheral blood and peritoneal exsudate were incubated with WGA and Con A (5-20 nm, Sigma; for 15 min - 96 hours, at 22 ° or 4°C). Macrophages contained gold label in heterogeneous vacuoles of medium or large size. Cells with lymphocytic morphology (with or without granules) showed binding at the surface which was reduced in favour of small, gold-marked vesicles with increasing incubation time. This course of endocytosis corresponds to the mentioned results in human cells (1). (1) Schumacher U., Wiedemann E., Steinmann G.G.: Wheat germ agglutinin uptake by cytotoxic lymphocytes. - Protoplasma 151 (124-127), 1989. (2) Harada K., Yamane H., Imai Y., Tsuji T., Toyoshima S., Osawa T. : Surface glycoprotein of human natural killer cells recognized by WGA. - Glycoconjugate J. 9 (198-203), 1992.
D.P12 ISOLATION OF A PARTIAL CODING SEQUENCE SYNTHASE FROM RAINBOW TROUT MACROPHAGES
FOR
NITRIC
OXIDE
L.J. Hardie ~, P. Grabowski 2, S. Ralston2, F. McGuigan 1, C.J. Secombes 1 ~Department of Zoology, University of Aberdeen, Aberdeen, Scotland, UK, and 2Department of Medicine and Therapeutics, University of Aberdeen Medical School, Aberdeen, Scotland, UK The enzyme responsible for the generation of nitric oxide radicals in mammalian species is NO synthase (NOS). In macrophages this enzyme exists in an inducible isoform (iNOS), which is present in activated macrophages after stimulation with cytokines and endotoxin. NO released from macrophages has been shown to be an important defence molecule, with potent antiparasitic, bactericidal and tumouricidal activities. To determine whether iNOS is conserved in teleosts, rainbow trout were challenged for 48h with an attenuated strain of Aeromonas salmonicida. Head kidney macrophages were isolated from these fish, the mRNA isolated and reverse transcribed to produce cDNA. Utilising two sets of primers constructed against conserved regions of mamalian iNOS forms, a nested PCR was performed. This yielded a 490 bp product which was reamplified and directly sequenced. The sequenced product (around 400 bp) shared 60-65% nucleotide and 70% amino acid identity to other (mammalian) iNOS sequences. Interestingly, the calmodulin binding site for this enzyme was not conserved. Nevertheless, this result strongly suggests that iNOS is present in teleosts and may have functional significance in disease resistance.
D.Pll UPTAKE
The Scientific and Social Program of the W h ISDCI Congress
OF GOLD-LABELED
LECTINS
( W G A , C O N A) B Y L E U C O C Y T E S
OF
T H E C A R P (CYPRINUS CARPIO L.) Bielek Edith Histological & Embryological Institute, University of Vienna, Schwarzspanierstr. 17, A-1090 Vienna, Austria In lymphocytes, the internalization of macromolecules and membrane components plays an important role in activation and regulation. Lectins binding to the surface glycoproteins and mediating different functions may be used as markers. The uptake of WGA (wheat germ agglutinin) by human cytotoxic lymphocytes was observed after light microscopic fluorescence labeling (1), as well as the preferential binding of WGA by natural killer cells (2). In the present study, binding and uptake of gold-labeled lectins were studied electron-microscopically in the carp. Leucocytes of peripheral blood and peritoneal exsudate were incubated with WGA and Con A (5-20 nm, Sigma; for 15 min - 96 hours, at 22 ° or 4°C). Macrophages contained gold label in heterogeneous vacuoles of medium or large size. Cells with lymphocytic morphology (with or without granules) showed binding at the surface which was reduced in favour of small, gold-marked vesicles with increasing incubation time. This course of endocytosis corresponds to the mentioned results in human cells (1). (1) Schumacher U., Wiedemann E., Steinmann G.G.: Wheat germ agglutinin uptake by cytotoxic lymphocytes. - Protoplasma 151 (124-127), 1989. (2) Harada K., Yamane H., Imai Y., Tsuji T., Toyoshima S., Osawa T. : Surface glycoprotein of human natural killer cells recognized by WGA. - Glycoconjugate J. 9 (198-203), 1992.
D.P12 ISOLATION OF A PARTIAL CODING SEQUENCE SYNTHASE FROM RAINBOW TROUT MACROPHAGES
FOR
NITRIC
OXIDE
L.J. Hardie ~, P. Grabowski 2, S. Ralston2, F. McGuigan 1, C.J. Secombes 1 ~Department of Zoology, University of Aberdeen, Aberdeen, Scotland, UK, and 2Department of Medicine and Therapeutics, University of Aberdeen Medical School, Aberdeen, Scotland, UK The enzyme responsible for the generation of nitric oxide radicals in mammalian species is NO synthase (NOS). In macrophages this enzyme exists in an inducible isoform (iNOS), which is present in activated macrophages after stimulation with cytokines and endotoxin. NO released from macrophages has been shown to be an important defence molecule, with potent antiparasitic, bactericidal and tumouricidal activities. To determine whether iNOS is conserved in teleosts, rainbow trout were challenged for 48h with an attenuated strain of Aeromonas salmonicida. Head kidney macrophages were isolated from these fish, the mRNA isolated and reverse transcribed to produce cDNA. Utilising two sets of primers constructed against conserved regions of mamalian iNOS forms, a nested PCR was performed. This yielded a 490 bp product which was reamplified and directly sequenced. The sequenced product (around 400 bp) shared 60-65% nucleotide and 70% amino acid identity to other (mammalian) iNOS sequences. Interestingly, the calmodulin binding site for this enzyme was not conserved. Nevertheless, this result strongly suggests that iNOS is present in teleosts and may have functional significance in disease resistance.