(44.192.112.123) 您好!臺灣時間:2021/03/09 00:09
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:董于瑄
研究生(外文):Yu-Hsuan Tung
論文名稱:鎘離子誘發類金屬硫蛋白於裂足海葵上的表現
論文名稱(外文):Cadmium induced expression of putative metallothioneins in sea anemones, Aiptasia pulchella
指導教授:陳俊宏陳俊宏引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:54
中文關鍵詞:金屬硫蛋白海葵
外文關鍵詞:cadmiummetallothioneinsea anemone
相關次數:
  • 被引用被引用:0
  • 點閱點閱:444
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
金屬硫蛋白 (Metallothionein, MT) 是一含有大量cysteine並且耐熱的小分子量蛋白質。這類蛋白質已經證實具有螯合¬二價重金屬的功用,除了可以調控必需重金屬鋅或銅的含量外,也可以在生物體內螯合非必需的重金屬如鎘或汞等,以減輕毒性,作為解毒機制的一環。從文獻知道,現存大部分物種從細菌到哺乳類動物皆發現有此類蛋白質,包括軟體、節肢及環節動物身上皆已証實有金屬硫蛋白的存在,然而於刺絲胞動物門的海葵至今是否存有金屬硫蛋白仍尚未被證實。本實驗中的動物,海葵Aiptasia pulchella 容易飼養並具有無性生殖的特性,可在實驗的過程中減少個體差異。經由實驗結果得知重金屬鎘除了會對海葵造成外觀上的傷害,也會進一步造成海葵部分累積較高量重金屬的組織脫落。鎘處理過的A. pulchella以immunoblotting可以偵測到三個耐熱的低分子量蛋白質,再以免疫組織染色可以更進一步看到金屬硫蛋白主要出現在海葵組織外胚層的部分,在重金屬鎘離子處理後明顯看到其被誘發表現的現象。雖然利用相近物種設計而來的核酸引子無法以RT-PCR在海葵身上找到確切的金屬硫蛋白序列,但是依據immunoblotting及免疫組織染色的結果,偵測到的蛋白質其分子量大小,耐熱特性以及表現模式,因此應可確認在海葵體內存在著金屬硫蛋白。

Metallothioneins (MTs) are cysteine rich, heat stable proteins with low molecular weight. They have been demonstrated that can chelate various divalent heavy metals. In addition to mediate homeostasis of essential metals Zn and Cu, MTs can bind with the nonessential metals, like Cd and Hg, to reduce their toxicities, which is recognized as a mechanism of detoxification. According to previous studies, MTs have been identified in a wide range of organisms, from bacteria to mammals. In invertebrate, MTs were proven existing in molluscs, crustaceans and annelids, but not in sea anemones. Sea anemone, Aiptasia pulchella, was chosen as the experimental organism in this study, because they are easy to be cultured in a laboratory system and have a life cycle with asexual reproduction, which reduce inter-individual variation. After exposed to cadmium, obvious morphological changes can be observed such as the shed tissues accumulated higher level of cadmium than normal tissues. From the results of immunoblotting, three heat stable, low molecular MT-like proteins were detected. Furthermore, the results of immunohistochemistry study revealed that the MTs expressed mainly in ectoderm and were induced after cadmium treatment. Although the exact MT sequence on A. pulchella was not obtained by using RT-PCR with primer combinations based on the immunobloting and immunohistochemistry results, the MT molecules were expressed in the rational location and their expression levels were induced after cadmium exposure. Therefore, it is strongly believed that MT molecules exist in A. pulchella.

口試委員會審定書 I
誌謝 II
Abstract IV
中文摘要 VI
Contents VII

1. Introduction
1.1. Heavy metals and cadmium 1
1.2. Cadmium pollution and toxicity 2
1.3. Protection mechanisms in animals 4
1.4. Characteristics of metallothioneins 4
1.5. Previous studies of vertebrate and invertebrate metallothioneins 6
1.6. Cnidarians and Aiptasia pulchella 8
1.7. Objective 9
2. Materials and Methods
2.1. Animals 11
2.2. Cadmium exposure 11
2.3. Measurement of cadmium bioaccumulation 11
2.4. RNA extraction and reverse transcription 12
2.5. Polymerase chain reaction (PCR) 13
2.6. Partial purification of metallothionein 13
2.7. SDS-PAGE and immunoblotting 14
2.8. Immunohistochemistry (IHC) 15
2.9. Statistic 17
3. Results
3.1. Morphological changes in A. pulchella during cadmium treatment 18
3.2. Bioaccumulation of cadmium in A. pulchella 18
3.3. Molecular analyses of metallothionein genes on A. pulchella 19
3.4. Detection of metallothioneins on A. pulchella by immunoblotting 20
3.5. Location of metallothioneins in A. pulchella 21
4. Discussion 23
5. References 29
Figures 41
Tables 52


Almedia, P., and L. Stearns. 1998. Political opportunities and local grassroots environmental movements: The case of Minamata. Society Problems. 45:37.
Amiard, J.C., C. Amiard-Triquet, S. Barka, J. Pellerin, and P.S. Rainbow. 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquatic Toxicology. 76:160-202.
Andersen, O., J.B. Nielsen, and P. Svendsen. 1988. Oral cadmium chloride intoxication in mice: effects of dose on tissue damage, intestinal absorption and relative organ distribution. Toxicology. 48:225-36.
Angerer, L., G. Kawczynski, D. Wilkinson, M. Nemer, and R. Angerer. 1986. Spatial patterns of metallothionein mRNA expression in the sea urchin embryo. Developmental biology. 116:543-547.
Barka, S., and J. Pavillon. 2000. Detoxification pathways and tissular localization of trace metals (copper, zinc, nickel, cadmium, silver and mercury) in a marine crustacean Tigriopus brevicornis (Muller). Study of the biomarker Metallothionein-like proteins, metal bioaccumulation and implication on higher trophic level transfer. Doctoral dissertation, Universite de Paris 06, Paris, France (in French with English abstract).
Barsyte, D., K.N. White, and D.A. Lovejoy. 1999. Cloning and characterization of metallothionein cDNAs in the mussel Mytilus edulis L. digestive gland. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology. 122:287-96.
Berthet, B., C. Mouneyrac, T. Perez, and C. Amiard-Triquet. 2005. Metallothionein concentration in sponges (Spongia officinalis) as a biomarker of metal contamination. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology. 141:306-13.
Binz, P., and J. Kagi. 1999. Metallothionein: molecular evolution and classification. Metallothionein IV:7-13.
Bouche, M.L., F. Habets, S. Biagianti-Risbourg, and G. Vernet. 2000. Toxic effects and bioaccumulation of cadmium in the aquatic oligochaete Tubifex tubifex. Ecotoxicology and Environmental Safety. 46:246-51.
Brouwer, M., D.R. Winge, and W.R. Gray. 1989. Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus. Journal of Inorganic Biochemistry. 35:289-303.
Bryan, G.M. 1976. Heavy metal contamination in the sea. In: Johnston R. (Eds.). Marine Pollution. Academic Press, New York, USA, pp. 185-302.
Chapman, G., J. Kay, and P. Kille. 1999. Structural and functional analysis of the rat metallothionein III genomic locus. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression. 1445:321-329.
Chomczynski, P and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform. Analytical Biochemistry. 162:156
Cuypers, A., M. Plusquin, T. Remans, M. Jozefczak, E. Keunen, H. Gielen, K. Opdenakker, A.R. Nair, E. Munters, T.J. Artois, T. Nawrot, J. Vangronsveld, and K. Smeets. 2010. Cadmium stress: an oxidative challenge. Biometals.
Davis, S.R., and R.J. Cousins. 2000. Metallothionein expression in animals: a physiological perspective on function. Journal of Nutrition. 130:1085-8.
Dhainaut-Courtois, N. 1988. Biological effects of three heavy metals (chromium, nickel and lead) in Nereis diversicolor. Oceanis. 14:423-433.
Dondero, F., L. Piacentini, M. Banni, M. Rebelo, B. Burlando, and A. Viarengo. 2005. Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation. Gene. 345:259-70.
Duffus, J.H. 2002. Effect of Cr (VI) exposure on sperm quality. Annals of Occupational Hygiene. 46:269-70.
Engel, D., and M. Brouwer. 1993. Crustaceans as models for metal metabolism: I. Effects of the molt cycle on blue crab metal metabolism and metallothionein. Marine Environmental Research. 35:1-5.
Eriksen, K., T. Andersen, J. Stenersen, and R. Andersen. 1990. Cytosolic binding of Cd, Cu, Zn and Ni in four polychaete species. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology. 95:111-115.
Fowler, B.A., C.E. Hildebrand, Y. Kojima, M. Webb. 1987. Nomenclature of metallothionein. Experientia Supplement. 52: 19-22.
Frazier, J.M. 1986. Cadmium-binding proteins in the mussel, Mytilus edulis. Environmental Health Perspectives. 65:39-43.
Ford, T.C. and J.M. Graham. 1991. An introduction to centrifugation. Bios Scientific Publishers Limited. Oxford.
Grant, A., K. Trompf, D. Seung, L. Nivison-Smith, H. Bowcock, H. Kresse, S. Holmes, J. Radford, and P. Morrow. 2010. Sub-cellular damage by copper in the cnidarian Zoanthus robustus. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 152:256-262.
Grassi, M., R. Tardent, and P. Tardent. 1995. Quantitative data about gametogenesis and embryonic development in Hydra vulgaris Pall.(Cnidaria, Hydrozoa). Invertebrate reproduction & development. 27:219-232.
Gupta, R., E. Gupta, B. Dhakal, A. Thakur, and J. Ahnn. 2004. Vitamin C and vitamin E protect the rat testes from cadmium-induced reactive oxygen species. Molecules and cells. 17:132-139.
Hamer, D.H. 1986. Metallothionein. Annual Review of Biochemistry. 55:913-51.
Harrison, P. and G. Waites. 1988. The Cassell Dictionary of Chemistry, Cassell, London.
Hogstrand, C. and C. Haux. 1991. Binding and detoxification of heavy metals in lower vertebrates with reference to metallothionein. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology Toxicology and Endocrinology. 100: 137-141.
Huang, C.I. 2007. The Induction of metallothioneins by zinc or cadmium in sea anemones, Aiptasia pulchella. Master thesis, Institute of Zoology, National Taiwan University.
Isani, G., G. Andreani, M. Kindt, and E. Carpene. 2000. Metallothioneins (MTs) in marine molluscs. Cellular and molecular biology (Noisy-le-Grand, France). 46:311.
Jarup, L., and A. Akesson. 2009. Current status of cadmium as an environmental health problem. Toxicology and Applied Pharmacology. 238:201-8.
Jarup, L., M. Berglund, C.G. Elinder, G. Nordberg, and M. Vahter. 1998. Health effects of cadmium exposure--a review of the literature and a risk estimate. Scandinavian Journal of Work, Environment and Health. 24 (3):240.
Kagi, J. 1993. Evolution, structure and chemical activity of class I metallothioneins: an overview. In: Suzuki KT, Imura N, Kimura M (Eds). Metallothionein III. Biological Roles and Medical Implications. Birkhauser Verlag, Berlin, pp. 29-56.
Kim, D.S., E.H. Lee, S.D. Yu, J.H. Cha, and S.C. Ahn. 2005. Heavy metal as risk factor of cardiovascular disease--an analysis of blood lead and urinary mercury. Journal of Preventive Medicine and Public Health. 38:401-7.
Kotsonis, F., and C. Klaassen. 1977. Toxicity and distribution of cadmium administered to rats at sublethal doses. Toxicology and Applied Pharmacology. 41:667-680.
Langston, W.J., M.J. Bebianno, and G.R. Hurt. 1998. Metal handling strategies in molluscs. In: Metal metabolism in aquatic environments. Springer, pp. 219-282.
Lawrence, J. 2010. Energetic Costs of Loss and Regeneration of Arms in Stellate Echinoderms. Integrative and Comparative Biology.
Lin, J., C.C. Tsai, W.K. Lai, and C.P. Chen. 1992. Pedal laceration in the sea anemone Aiptasia sp. (Anthozoa: Actiniaria). Chinese Bioscience. 35 (1):33-41.
Mackay, E.A., J. Overnell, B. Dunbar, I. Davidson, P.E. Hunziker, J.H. Kagi, and J.E. Fothergill. 1993. Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis. European Journal of Biochemistry. 218:183-94.
Margoshes, M., and B. Vallee. 1957. A cadmium protein from equine kidney cortex. Journal of the American Chemical Society. 79:4813-4814.
Miles, A.T., G.M. Hawksworth, J.H. Beattie, and V. Rodilla. 2000. Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Critical Reviews in Biochemistry and Molecular Biology. 35:35-70.
Morgan, M.B., S.E. Edge, and T.W. Snell. 2005. Profiling differential gene expression of corals along a transect of waters adjacent to the Bermuda municipal dump. Marine Pollution Bulletin. 51:524-33.
Nawrot, T.S., E. Van Hecke, L. Thijs, T. Richart, T. Kuznetsova, Y. Jin, J. Vangronsveld, H.A. Roels, and J.A. Staessen. 2008. Cadmium-related mortality and long-term secular trends in the cadmium body burden of an environmentally exposed population. Environmental Health Perspectives. 116:1620-8.
Nemer, M., E. Travaglini, E. Rondinelli, and J. D. Alonzo. 1984. Developmental regulation, induction, and embryonic tissue specificity of sea urchin metallothionein gene expression. Developmental biology. 102:471-482.
Nriagu, J.O., and J.M. Pacyna. 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature. 333:134-9.
Olsson, M.H., U. Ryde, and B.O. Roos. 1998. Quantum chemical calculations of the reorganization energy of blue-copper proteins. Protein Science. 7:2659-68.
Olsson, P., M. Zafarullah, R. Foster, T. Hamor, and L. Gedamu. 1990. Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri. European Journal of Biochemistry. 193:229-235.
Otvos, J.D., and I.M. Armitage. 1980. Structure of the metal clusters in rabbit liver metallothionein. Proceedings of the National Academy of Science of the United States of America. 77:7094-8.
Park, J.D., Y. Liu, and C.D. Klaassen. 2001. Protective effect of metallothionein against the toxicity of cadmium and other metals. Toxicology. 163:93-100.
Philp, R.B. 1999. Cadmium content of the marine sponge Microciona prolifera, other sponges, water and sediment from the eastern Florida panhandle: possible effects on Microciona cell aggregation and potential roles of low pH and low salinity. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology. 124:41-9.
Quaife, C.J., S.D. Findley, J.C. Erickson, G.J. Froelick, E.J. Kelly, B.P. Zambrowicz, and R.D. Palmiter. 1994. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry. 33:7250-9.
Rand, G. M., P. G. Wells, L. S. McCarty. 1995. Introduction to aquatic toxicology. In Fundamentals of Aquatic Toxicology, G. M. Rand (Ed.), Taylor and Francis, Washington, DC.
Riek, R., B. Precheur, Y. Wang, E.A. Mackay, G. Wider, P. Guntert, A. Liu, J.H. Kagi, and K. Wuthrich. 1999. NMR structure of the sea urchin (Strongylocentrotus purpuratus) metallothionein MTA. Journal of Molecular Biology. 291:417-28.
Roesijadi, G. 1992. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquatic Toxicology. 22:81-114.
Roesijadi, G., and B.A. Fowler. 1991. Purification of invertebrate metallothioneins. Methods in Enzymology. 205:263-73.
Roesijadi, G., K.M. Hansen, and M.E. Unger. 1996. Cadmium-induced metallothionein expression during embryonic and early larval development of the mollusc Crassostrea virginica. Toxicology and Applied Pharmacology. 140:356-63.
Roesijadi, G., and P. Klerks. 1989. Kinetic analysis of cadmium binding to metallothionein and other intracellular ligands in oyster gills. Journal of Experimental Zoology. 251:1-12.
Roeva, N., A. Sidorov, and Y. Yurovitskii. 1999. Metallothioneins, proteins binding heavy metals in fish. Biology Bulletin. 26:617-622.
Sato, M., and I. Bremner. 1993. Oxygen free radicals and metallothionein. Free Radical Biology and Medicine. 14:325-37.
Schroder, H., K. Shostak, V. Gamulin, M. Lacorn, A. Skorokhod, V. Kavsan, and W. Muller. 2000. Purification, cDNA cloning and expression of a cadmium-inducible cysteine-rich metallothionein-like protein from the marine sponge Suberites domuncula. Marine ecology Progress series. 200:149-157.
Schwartz, G.G., and I.M. Reis. 2000. Is cadmium a cause of human pancreatic cancer? Cancer Epidemiology Biomarkers and Prevention. 9:139-45.
Smith, L. 1995. Effects of limb autotomy and tethering on juvenile blue crab survival from cannibalism. Marine ecology progress series. Oldendorf. 116:65-74.
Steinebach, O.M., and H.Th. Wolterbeek. 1994. Role of cytosolic copper, metallothionein and glutathione in copper toxicity in rat hepatoma tissue culture cells. Toxicology. 92:75-90.
Stone, H.C., S.B. Wilson, and J. Overnell. 1986. Cadmium-binding proteins in the scallop Pecten maximus. Environmental Health Perspectives. 65:189-91.
Sullivan, J.C, A.M. Reitzel, J.R. Finnerty. 2006. A High Percentage of Introns in Human Genes Were Present Early in Animal Evolution: Evidence from the Basal Metazoan Nematostella vectensis. Genome Informatics. 17: 219–229.
Taylor, D. 1983. The Significance of the Accumulation of Cadmium by Aquatic Organisms. Ecotoxicology and Environmental Safety. 7:33-42.
Templeton, D.M., and M.G. Cherian. 1991. Toxicological significance of metallothionein. Methods in Enzymology. 205:11-24.
Thevenod, F., and J. Friedmann. 1999. Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K+-ATPase through proteasomal and endo-/lysosomal proteolytic pathways. The Federation of American Societies for Experimental Biology Journal. 13:1751.
Vangronsveld, J., F. Van Assche, and H. Clijsters. 1995. Reclamation of a bare industrial area contaminated by non-ferrous metals: in situ metal immobilization and revegetation. Environmental Pollution. 87:51-9.
Vasak, M. 1991. Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods in Enzymology. 205:452-8.
Venuti, J., and K. Edds. 1986. Calmodulin and caimodulin-binding proteins in the morphological transformation of sea urchin coelomocytes. Cell Motility and the Cytoskeleton. 6:604-619.
Viarengo, A., B. Burlando, N. Ceratto, and I. Panfoli. 2000. Antioxidant role of metallothioneins: a comparative overview. Cellular and Molecular Biology (Noisy-le-grand, France). 46:407-17.
Viarengo, A., M. Moore, M. Pertica, G. Mancinelli, G. Zanicchi, and R. Pipe. 1985. Detoxification of copper in the cells of the digestive gland of mussel: the role of lysosomes and thioneins. Science of the Total Environment. 44:135-145.
Viarengo, A., and J. Nott. 1993. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comparative Biochemistry and Physiology Part C: Comparative and Pharmacology. 104:355-372.
Vidal, D.E., and A.J. Horne. 2003. Mercury toxicity in the aquatic oligochaete Sparganophilus pearsei. II: Autotomy as a novel form of protection. Archives of Environmental Contamination and Toxicology. 45:462-7.
Waalkes, M.P. 2003. Cadmium carcinogenesis. Mutation Research. 533:107-20.
Wang, J.T., Y.J. Chou, J.H. Chou, C.A. Chen, and W.M. Chen. 2008. Tenacibaculum aiptasiae sp. nov., isolated from a sea anemone Aiptasia pulchella. International Journal of Systematic and Evolutionary Microbiology. 58:761-6.
Weber, J., M. Klug, and P. Tardent. 1987. Detection of high concentration of Mg and Ca in the nematocysts of various cnidarians. Cellular and Molecular Life Sciences. 43:1022-1025.
Weeks, M.E. 1968. Discovery of the Elements. Journal of Chemical Education. pp. 502-508.
Wicksten, M.K. 1989. Why Are There Bright Colors in Sessile Marine Invertebrates? Bulletin of Marine Science, 45(2):519-530.
Williams, K., D. Green, D. Pascoe, and D. Gower. 1986. The acute toxicity of cadmium to different larval stages of Chironomus riparius (Diptera: Chironomidae) and its ecological significance for pollution regulation. Oecologia. 70:362-366.
Winge, D.R., B.L. Geller, and J. Garvey. 1981. Isolation of copper thionein from rat liver. Archives of Biochemistry and Biophysics. 208:160-6.
Wu, J.P. 2005. Toxic effects of cadmium and zinc on white shrimp, Litopenaeus vannamei (Boone, 1931). Doctoral dissertation, Institute of Zoology, National Taiwan University (in Chinese with English abstract).
Zaroogian, G., and E. Jackim. 2000. In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology. 127:251-61.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔