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標題: 應用重金屬抗性相關蛋白於汞之生物復育研究
Application of heavy metal resistance related proteins for mercury bioremediation
作者: 林國興
Lin, Kuo-Hsing
關鍵字: metallothionein

organomercurial lyase
出版社: 生命科學系所
引用: 鄒曉薇 (2009) 利用X-ray繞射法解析轉位子TnMERI1汞抗性操作組轉錄調控基因子MerR1結構與分析汞離子還原酶(MerA)及有機汞裂解酶(MerB1、MerB2、MerB3)之交互作用. Allen P (1994) Mercury accumulation profiles and their modification by interaction with cadmium and lead in the soft tissues of the cichlid Oreochromis aureus during chronic exposure. Bull Environ Contam Toxicol 53:684-692 Amyot M, Southworth G, Lindberg SE, Hintelmann H, Lalonde JD, Ogrinc N, Poulain AJ, Sandilands KA (2004) Formation and evasion of dissolved gaseous mercury in large enclosures amended with HgCl2. Atmos Environ 38:4279-4289 Andrews GK (1990) Regulation of metallothionein gene expression. Prog Food Nutr Sci 14:193-258 Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59:95-104 Aschner M, Walker SJ (2002) The neuropathogenesis of mercury toxicity. 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摘要: 汞及其衍生物已經知道為對於人類的腦及神經並會導致神經退化疾病傷害的神經毒性物質,汞非常容易藉由水域中的底泥中微生物轉化成甲基汞(methylmercury) 經由食物鏈的方式達到生物放大的效果。由過去,例如1950年代日本水俁地區與1970年代伊拉克,都發現因為藉由食物鏈的關係而累積大量甲基汞造成中毒現象。金屬硫基蛋白(metallthionein,MT)是一低分子量,高cysteine含量的膜內蛋白,對金屬離子具有高度親和力,可藉由對 Cd2+、Hg2+ 或 MeHg+的結合達到減低對生物體毒性的能力。另外,MT也發現具有清除自由基 (free radicals) 的能力。本文將人類、老鼠與吳郭魚的MT利用基因工程方式將其表現在E coli. 胞內或是與外膜蛋白OmpC螯合以表現至外膜上。在暴露於含有汞或是自由基的環境下,發現表現MT至外膜上的細菌都比表現MT於細胞質中的細菌至少高出大約10%的清除能力。然而,在所有的三個MT物種中,實驗結果也顯示吳郭魚的MT蛋白具有最好的去除汞及自由基的能力。另外,實驗利用caboxy-H2DCFA的化學物質來觀察細菌體內的自由基多寡。在利用鎘誘導產生氧化壓力的實驗下,顯示當細胞所產生的MT可以結合金屬的能力越強,則胞內產生的自由基數目則越少。 此外,篩選自水俁灣之Bacillus megaterium MB1菌株之汞抗性基因組中的merB3 基因可表現廣泛性抗有機汞的能力的有機汞裂解酶,並且已知對於甲基汞和乙酸苯汞 (phenylmercuric acetate)都具有裂解的能力。實驗發現利用純化後的MerB1、2、3 蛋白進行體外實驗,結果顯示MerB1和3都具有分解甲基汞和乙酸苯汞的能力,但是MerB2卻只對乙酸苯汞有些微的裂解能力。經由注射merB3基因並在肌肉專一表現有機汞裂解酶的轉殖斑馬魚在毒性測試下,發現轉殖魚曝露在甲基汞下LC50為404nM而野生種為376nM。若為乙酸苯汞,野生種L50則是200nM而轉殖魚為237nM。結果呈現轉殖斑馬魚對於有機汞抗性有上升的趨勢。但是轉殖魚與野生種之胚胎在不同發育時期開始曝露於不同有機汞及濃度對尾鰭形成影響的實驗中,觀察顯示轉殖魚尾鰭發育在不同濃度的有機汞下,畸形狀況與野生種並無相當的明顯差異,因此無法從形態上確認抗性的能力。
Mercury and its derivates is known toxic to human beings as well as neurotoxicity to damage brain and led to neurondegeneration disease. In aquatic system, mercury is easily transferred to methylmercury by bacteria in the sediment. As some poisoned case in the past, such as Minamata in Japan in 1950s and Iraq in 1970, it is identified that methylmercury were taken in through food chain. Metallothioneins (MTs) are low-molecular-weight, cysteine-rich intracellular proteins with high affinity to toxic metal cations for detoxification, such as Cd2+, Hg2+ and MeHg+ and also possess the ability of scavenging reactive oxygen species. The tilapia, human and mouse MTs were expressed either in cytosol or fusion to outer membrane protein (OmpC) in the recombinant E. coli. Exposure to mercury or free radicals shows that outer-membrane-target ones had 10 to 20 % higher sequestering ability than cytosolically ones. In scavenging 2,2-azinobis(3-ethylbenzothiazoline-6 -sulfonic acid) diammonium salt free radicals, the outer membrane MTs had 10% higher scavenging ability than cytosolic MTs. Among three MTs, tilapia MT presents best activity of adsorbing mercury and scavenging radicals. Moreover, the caboxy-H2DCFDA was used to detect reactive oxygen species. It is observed that the oxidative stress decrease was dependent on cadmium binding ability as higher metal bonding ability led to lower oxidative stress induced by cadmium. Methylmercury is frequently found in aquatic system and could be amplified through food chain. On the other hand, the organomercurial lyase merB3 gene of Bacillus megaterium MB1 is known to have board spectrum organomercurials resistance enzymes. The purified MerB3 was characterized in vitro with the presence of phenylmercuric acetate (PMA) and methylmercury chloride (MMC) and present similar catalytic activity to MerB1 whereas, MerB2 did not possess the ability to degrade the two organomercurials. As introducing merB3 gene to zebrafish, the LC50 of wild type and transgenic zebrafish in MMC were 376nM and 404nM, respectively, while in PMA were 200nM and 237nM. The transgenesis show better resistance ability than wild type. However, the phenotype of teratogenic effects on tail was no obvious difference for both zebrafish.
其他識別: U0005-2308201022053900
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