open access
메뉴
ISSN : 1225-3480
Nitric oxide (NO) is an important intra-intercellular signaling molecule that regulates many physiological processes and participates in the development some pathological conditions in animals. In this study, we compared different methods for determining NO concentration in the hemocytes of Manila clam Ruditapes philippinarum. For measuring the intracellular NO levels, we used the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA), and the quantification methods that were compared were based on image analysis, spectrophotometry, and flow cytometry. NO concentration could be determined using all the 3 methods, and the concentration varied significantly depending upon the presence of NO regulators in the hemocytes; NO concentration increased in the presence of L-arginine, while it decreased in the presence of N-nitro-L-arginine methyl ester. In particular, it is found that estimation of NO using a flowcytometry is more economical, reliable and accurate compared to image analysis and spectrophotometry. Accordingly we believe that determining NO concentration by using flowcytometry will be useful in evaluating physiological and pathological conditions in marine bivalves.
(2001). Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study. FEBS Letters, 506, 185-190. 10.1016/S0014-5793(01)02880-0.
(2000). Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Letters, 468, 89-92. 10.1016/S0014-5793(00)01203-5.
(1998). The role of nitric oxide in inflammation and immunity. Arthritis and Rheumatism, 41, 1141-1151. 10.1002/1529-0131(199807)41:7<1141::AID-ART2>3.0.CO;2-S.
Image J User Guide - IJ 1.46r.
(2008). Immunotoxic effects on freshwater mussels of a primary-treated wastewater before and after ozonation: a pilot plant study. Ecotoxicology and Environmental Safety, 69, 366-373. 10.1016/j.ecoenv.2007.10.027.
(2005). Inflammatory properties of municipal effluents to Elliptio complanata mussels - lack of effects from anti-inflammatory drugs. Comparative Biochemistry and Physiology, 141(C), 332-337.
(2002). cDNA cloning, characterization and gene expression of nitric oxide synthase from the silkworm. Bombyx mori. Insect Molecular Biology, 11, 257-265. 10.1046/j.1365-2583.2002.00333.x.
(2011). Involvement of nitric oxide in the in vitro interaction between Manila clam, Ruditapes philippinarum, hemocytes and the bacteriumVibrio tapetis. Fish and Shellfish Immunology, 31, 1137-1141. 10.1016/j.fsi.2011.10.008.
(2006). Cadmium induced endothelial dysfunction: Consequence of defective migratory pattern of endothelial cells in association with poor nitric oxide availability under cadmium challenge. Cell Biology International, 30, 427-438. 10.1016/j.cellbi.2006.02.002.
(1996). Nitric oxide synthase and nitrite production in human spermatozoa: evidence that endogenous nitric oxide is beneficial to sperm motility. Molecular Human Reproduction, 2, 873-878. 10.1093/molehr/2.11.873.
(2009). Estimation of nitric oxide as an inflammatory maker in periodontitis. Journal of Indian Society of Periodontology, 13, 75-78. 10.4103/0972-124X.55842.
(1998). Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Letters, 427, 263-266. 10.1016/S0014-5793(98)00440-2.
(1987). Nitric oxide release accounts for the biological activity of endothelium derived relaxing factor. Nature, 327, 524-526. 10.1038/327524a0.
(2003). Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes. Thrombosis Research, 110, 117-126. 10.1016/S0049-3848(03)00312-8.
(2003). Nitrite oxide production by carpet shell calm (Ruditapes decussatus) hemocytes. Developmental and Comparative Immunology, 27, 197-205. 10.1016/S0145-305X(02)00098-8.
(2004). iNOS-mediated nitric oxide production and its regulation. Life Sciences, 75, 639-653. 10.1016/j.lfs.2003.10.042.
(2002). Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams. Diseases of Aquatic Organisms, 48, 221-231. 10.3354/dao048221.
(2009). Morphological and molecular effects of Vibrio splendidus on hemocytes of softshell clams. Mya arenaria. Journal of Shellfish Research, 28, 751-758. 10.2983/035.028.0403.
(1997). Hemocyte aggregation in the oyster Crassostrea gigas: In vitro measurement and experimental modulation by xenobiotics. Comparative Biochemistry and Physiology Part A: Physiology, 118, 705-712. 10.1016/S0300-9629(97)00017-0.
Activity of endothelial nitric oxide synthesis: substrate, modulators and products.
(2001). Nitric oxide and the immune response. Nature Immunology, 2, 907-916. 10.1038/ni1001-907.
Molluscan immunobiology. In; The Mollusca, Vol. 5, Physiology.
(2007). Methods to detect nitric oxide and its metabolites in biological samples. Free Radical Biology and Medicine, 43, 645-657. 10.1016/j.freeradbiomed.2007.04.026.
(1995). Inducible nitric oxide synthase in inflammation. The Histochemical Journal, 27, 777-784. 10.1007/BF02388303.
(2006). Nitric oxide and inflammation. Inflammation and Allergy Drug Targets, 5, 115-119. 10.2174/187152806776383143.
