熱逆境後小球藻粒腺體活性的衰退/抗菌多胜肽對生長中大腸桿菌的影響

當細胞面對極端的環境時，其生理與型態上都會發生改變。從之前的研究中發現，小球藻在經過46.5℃一小時熱逆境後，分別在光照或黑暗中繼續培養，其細胞核內的DNA會聚集，並且在光照培養下可觀察到葉綠素的降解和部份DNA的分解，所以懷疑熱逆境處理後的小球藻可能會走向程序性的細胞死亡。為了更進一步了解細胞內的變化狀況，本實驗首先觀察加熱46.5℃時細胞核內DNA的早期變化(一個小時內)，並且利用細胞活性測試，觀察整體或個別細胞的粒線體琥珀酸脫氫酶的酵素活性；令人驚訝的，在加熱後暗處理的細胞中，其細胞活性逐漸降低，似乎逐漸步入死亡，而加熱後光照處理的細胞，則有部份逃過死亡，不但恢復了酵素活性，細胞重新開始分裂，也重新的產生葉綠素螢光，其中的修補機制值得未來更深入的探討。由於病原菌抗藥性日漸嚴重，近年來開始著重於有關於抗菌蛋白(antimicrobial peptides)(AMPs)的研究，且有許多的AMPs已經在動物、植物或是昆蟲上發現。本實驗由非洲爪蛙蟾蜍(African clawed frog Xenopus laevis)表皮所分泌的抗菌蛋白：magainin 2(MG2)為基礎，改變其親水性胺基酸的分布或是螺旋的數目，並藉由大腸桿菌的生長曲線觀察所造成的影響，結果發現表現MG2本身對大腸桿菌的生長並沒有太大的影響，反而是較短但具有MG2胺基端的MG3對菌的生長有抑制的作用，其中的原理仍需要更深入的探討。

關鍵字

小球藻；熱逆境； MTT 實驗；共軛焦顯微鏡；粒線體；抗菌多胜肽；生長曲線

並列摘要

When cell exposed to extreme environment, both their physiology and morphology change accordingly. In previous studies, we found that Chlorella pyrenoidosa exposed to heat stress (46.5℃) for one hour and cultured again under light or dark environment., their nuclear DNA would be condensed. If recultured under light, both chlorophylls and DNA would degrade. We thus speculated that heat stress plus readtirition might lead chlorella to go through a process of programmed cell death. In order to understand more about this process, we tried to detect the early DNA changes after heat stress by confocal microscopy and measured mitochondrial cell activity by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Surprisingly, If re-culture was done in the dark, cells’ activity would reduce. Continuously, leading cells to death. On the other hand, under continuous illumination some cell would escape from death, and repaired normal chlorophyll fluorescence and cell activity. This repair system certainly is worthy to study further. Because antibiotic resistances of pathogens are growing worse these day , research about antimicrobial peptides (AMPs) has become more important. Many AMPs have bean discovered in animals and plants, insects. In this study, was focused on magainin 2(MG2), an AMP secreted from African clawed frog Xenopus laevis. We changed MG2’s amino acid sequence or the number of helix number, and tested their effect on Escherichia coli’s growth. Our data indicate no change in E.coli’s growth curve, when MG2 was expressing. However, expression of MG3, which lacked the C-terminal helix of MG2, inhibited the growth of E.coli. The mechanisms behind requires further study.

並列關鍵字

chlorella ； heat stress ； MTT assay ； confocal microscopy ； mitochondria ； antimicrobial peptides ； growth curve

參考文獻

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熱逆境後小球藻粒腺體活性的衰退/抗菌多胜肽對生長中大腸桿菌的影響

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