| 1. To study nitric oxide production and nitric oxide synthase activity in the organizations of Epinephelus malabaricus. Griess Reagent was used to determinate the nitric oxide (NO) production, and the method of chemical colorimetry was used to measure the nitric oxide synthase (NOS) activity. Inducible NOS (iNOS) activity was highest in the head-kidney, followed by the muscle, liver, spleen, intestine, stomach, pancreas, eye, brain, heart, sill and kidney. The constitutive NOS (cNOS) activity was highest in the brain, followed by the eye, intestine, liver, stomach, muscle, head-kidney, sill, pancreas, kidney, heart and spleen. The spleen was found to have the highest level of NO production, followed by the liver, head-kidney, kidney, heart, pancreas, intestine, brain, eye, stomach, sill, muscle and serum.2. Immunohistochemical localization SP (streptavidin-perosidase) method and NADPH-diaphorase (nicotinamide adenine dinucleotide hydrogen phosphate-diapho-rase) staining were used to study distribution and localization of neuronal NOS (nNOS) in the organizations of Epinephelus malabaricus. The results of enzyme immunohistochemical localization showed that the positive results of nNOS were located in neurons of the diencephalons. in neurons and nerve fibers of brain and mesencephalon. in granular cells, nerve fibers purkinje cells of the cerebellum; in the retina, layer of cones and rods, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, layer of ganglion cell and layer of optic nerve fibers; in the kidney, proximal convoluted tubule and distal convoluted tubule; in the head kidney, anterior interrenal tissues and reticular cells; in the heart, nerve fibres and myocardial fibers; in the spleen, red cells of splenic sinusoids and the reticular cells of splenic cord; in the intestine, striated border, neurons and basement membrane; in the liver, liver cells and central vein; muscle fibres of the muscle; in the gill, epithelial cells of the filament, nerve fibers run along vein blood and red cells. The results of NADPH-d histochemical staining showed that NOS were distributed in neurons of the diencephalons, neurons and nerve fibers of brain and mesencephalon, granular cells, nerve fibers purkinje cells of the cerebellum; in the retina. layer of cones and rods, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, layer of ganglion cells and layer of optic nerve fibers; in the kidney, proximal convoluted tubules, distal convoluted tubules, nerve fibres and vessel wall; in the head kidney, anterior interrenal tissues, nerve fibers of arteries, neurons and reticular cells; in the heart, nerve fibers and myocardial fibers; the red cells and the reticular cells of the spleen; in the intestine, striated border, neurons and myenteric nerve fibers; in the liver, liver cells, nerve fibers, kupffer cells, interlobular artery, interlobular vein and interlobular bile duct; muscle fibres of the muscle; in the gill, epithelial cells of the filament, nerve fibers run along vein blood, chloride cells and red cells. Western Blot was used to analysis protein molecular weight of nNOS in organizations, the results show that there were three belts in the brain, eye, head kidney, kidney, heart, gill and stomach, respectively, corresponding molecular weight were 30 kD,120 kD and 80 kD. However, there were two belts in the intestine, spleen, liver, pancreas and muscle, respectively, corresponding molecular weight were 120 kD and 80 kD.3. To study the effect of irritants about nitric oxide production and nitric oxide synthase activity in the serum and head kidney of Epinephelabaricus. Irritants include:lipopolysaccharide (LPS), zymosan and Aeromonas hydrophila. The macrophages of head kidney were cultured in vitro, to study NO production and iNOS activity, the macrophages of head kidney were stimulated by LPS, the results show that iNOS of macrophages could be induced by LPS, a large amounts of NO could be catalyzed by iNOS. however, NG-minomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine methylester (L-NAME) could inhibit the production of NO. The most of iNOS activity appeared at 12 hour after stimulated, the most of NO production appeared at 24 hour after stimulated. To study NO production and iNOS activity in the serum and head kidney, zymosan and Aeromonas hydrophila were injected in abdominal cavity of Epinephelus malabaricus, the results show that the most of iNOS activity also appeared at 12 hour after stimulated, the most of NO production also appeared at 24 hour after stimulated. The study proved that growth of iNOS activity and increase of NO production had the same tendency in vivo and in vitro, growth of iNOS activity and increase of NO production had the same timeliness in vivo and in vitro. 4. Immunohistochemical localization SP method and NADPH-diaphorase staining were used to study distribution and localization of inducible NOS (iNOS) in the organizations of Epinephelus malabaricus. The results of enzyme immunohistochemical localization showed that iNOS were distributed in neurons and nerve fibers of the brain, purkinje cells of the cerebellum; in the retina, layer of cones and rods, outer plexiform layer, inner nuclear layer, inner plexiform layer, layer of ganglion cells and layer of optic nerve fibers; in the kidney, proximal tubules, distal convoluted tubules and macrophages; in the head kidney, anterior interrenal tissues, macrophages and neutrophils; myocardial fiber of the heart; in the spleen, the macrophages, neutrophils and lymphocytes; in the intestine, the striated border and macrophages; muscle fibres of the muscle; in the liver, liver cells and kupffer cells; in the gill, nerve fibers, epithelial cells and red cells. The results of NADPH-d histochemical staining showed that NOS were distributed in neurons and nerve fibers of the brain, purkinje cells of the cerebellum; in the retina, NOS were distributed in the cone and rod layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, the network layer, ganglion cell layer and optic nerve fiber layer; in the kidney, the proximal tubules, distal convoluted tubules and macrophages; in the head kidney, anterior interrenal tissues, macrophages and neutrophils; myocardial fiber of the heart; in the spleen, macrophages. neutrophils and lymphocytes; striated border and macrophages of the intestine; in muscle fibres of muscle; liver cells and kupffer cells of the liver; in the gill, nerve fibers, epithelial cells and chloride cells. Western Blot was used to determine protein molecular weight of iNOS in organizations, the results show that there were two belts in the brain, eye, muscle, gill, head kidney, stomach, liver, spleen, kidney and intestine, respectively,120 kD and 80 kD. There was one belt in the heart,80 kD. however, there was no belt appeared in the pancreas. |
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