Baicalein-induced apoptosis via endoplasmic reticulum stress through elevations of reactive oxygen species and mitochondria dependent pathway in mouse-rat hybrid retina ganglion cells (N18)

[[abstract]]Studies were designed to investigate the effects of baicalein on mouse-rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, reactive oxygen species (ROS), cytoplasmic Ca2+, mitochondrial membrane potential (MMP), apoptosis induction, and caspases-3 activity were examined by flow cytometric assay. Apoptosis-associated proteins such as p53, Bax, Bcl-2, cytochrome c, and caspase-3 were examined by Western blot. We demonstrated the increase in the levels of p53, Bax, and cytochrome c and decrease in the level of Bcl-2, which are associated with the induction of apoptotic cell death after 24 h treatment with baicalein in N18 cells. Baicalein induced an increase in the cytoplasmic levels of ROS and Ca2+ in 1 h and reached their peak at 3 h, and thereafter a loss of MMP by flow cytometry. We also demonstrated a release of the cytochrome c from mitochondria into cytosol and an activation of caspase-3, which led to the occurrence of apoptosis in N18 cells treated with baicalein by Western blot. Pretreatment was conducted with BAPTA (intracellular calcium chelator) in baicalein-treated cells, the decline of MMP was recovered, and the increase in the level of cytoplasmic Ca2+ was suppressed, and the proportion of apoptosis was also markedly diminished. In conclusion, our data suggests that oxidative stress and cellular Ca2+ modulates the baicalein-induced cell death via a Ca2+-dependent mitochondrial death pathway in N18 cells

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Ultrastructure of jelly fig achenes and purification, characterization, and antifungal activity analysis of a chitinase in the pericarp of achenes

愛玉為台灣特有種植物，愛玉凍風行於本地約有兩百年之久，但其間的研究，除了解開愛玉凝凍的原理以及一些栽種方法的改良之外，尚無更深一層的研究與探討；所以，本論文希望透過對愛玉基礎之研究，俾能找出愛玉其他的有用成份與其利用價值。 對於愛玉凍的凝膠，其原理雖已知曉，但在愛玉瘦果內究竟是從何而來的果膠與果膠甲基酯酶相互作用，使產生低甲氧基的果膠與愛玉瘦果本身的鈣等陽離子鍵結形成固態的愛玉凍，以及其形態與結構的瞭解，可說仍無所知曉。本研究利用顯微鏡檢技術、酵素免疫分析與免疫金原子標幟，將愛玉瘦果的形態及細微結構並瘦果果皮蛋白質的所在位置一一揭露出來：愛玉瘦果果皮僅由三至四層的活細胞層與約五層的死細胞層所構成；一大細胞層與一小細胞層的種皮內、外層具有獨特的自身色素；雙子葉植物種子的胚乳層與其內胚的所有種子細胞蘊含豐富的蛋白質體與油體；果膠甲基酯酶及幾丁質酶存在於愛玉瘦果果皮內的活細胞細胞質中，且以果膠甲基酯酶的含量較高；果膠主要位於種子内胚乳層的細胞壁上，因此，當將愛玉瘦果浸入水中搓洗，種子內大量的果膠流入水中，被果皮細胞中流出的果膠甲基酯酶切除甲氧基，形成低甲氧基的果膠，而與水中或愛玉瘦果中的陽離子例如鈣離子鍵結，而後凝結成凍。 本研究證實愛玉瘦果的果皮中含有一酸性幾丁質酶，在酸性的環境下皆具有酵素活性，其優異的生化特性與穩定結構，使之能耐高溫60℃達數小時之久，酵素活性不減，儲存於4℃低溫下近一年其酵素活性仍超過八成；又愛玉幾丁質酶針對含有幾丁質的植物病原真菌之細胞壁作用，可以將水果倉儲病原菌-炭疽病菌(Colletotrichum gloeosporioides)之病原孢子殺死，以防止成熟的水果表面產生變黑腐爛的病徵，對於蓮霧果腐病的病原菌(Rhizopus stolonifer)與十字花科蔬菜立枯絲核病菌(Rhizoctonia solani)菌絲之生長，亦有很好的抑制效果；此外對於番茄葉黴病菌(Fulvia fulva)及十字花科黑斑病(Alternaria brassicicola) 孢子之發芽，也有不錯的抑制功效；綜合上述研究成果，發現愛玉幾丁質酶具有耐熱與抗黴的特性，頗有研發成為防黴劑的潛能。 關鍵詞：愛玉瘦果、幾丁質酶、病原真菌、耐熱、果皮。Ultrastructure of jelly fig achenes and purification, characterization, and antifungal activity analysis of a chitinase in the pericarp of achenes Jelly fig (Ficus awkeotsang Makino) is a unique plant growing in Taiwan. There are no further researches and discussions about the jelly fig except for a clue to congealing of jelly curd and some improvement of planting methods during that time. There is also no morphological and structural information with regard to jelly fig achenes. This research focuses on the basic studies of jelly fig. Although the congealed mechanism of jelly curd has been discovered, it is still unknown where the gelled pectin and pectin methylesterase are present in the achene. Detail structures of the jelly fig achene and subcellular locations of its pericarpial proteins are still unclear. Therefore, this research utilized microscopic observation, enzyme immunoassay, and immunogold labeling to reveal the unsolved questions. The pericarp of jelly fig achene has only 3-4 layers of living cells and about five layers of death cell's conformations. A large cell layer and a small cell layer compose of the outer and inner layers of seed coat, which has its own unique pigment. Like other dicotyledons, an embryo and an endosperm exist in the seed. These cells contain abundant protein bodies and oil bodies. Pectin methylesterase and chitinase are present in the cytoplasm of live pericarpial cells. The content of pectin methylesterase is more than that of chitinase. The pectin lies mainly on the cell wall of the endosperm in the seed. Water extract of jelly fig achenes contains the pericarpial pectin methylesterase and endospermous pectin. The interaction between pectins and pectin methylesterases produces the low methoxyl pectins and then these pectins link with cations, to form the solid state of jelly curd. A chitinase was found in the pericarpial extract of jelly fig achene. This chitinase possess excellent biochemical characteristics and stable structure; it maintains fully activity after treated at 60 degrees centigrade for several hours. Most plant pathogens contain chitin in their cell walls. The spore germination of Colletotrichum gloeosporioides, the major postharvest pathogen of mango, papaya and many other fruits in Taiwan, was inhibited and killed by this chitinase. In addition, the spore germination of Fulvia fulva, and Alternaria brassicicola was also suppressed by the chitinase. The mycelial growth of Rhizoctonia solani AG-4 and Rhizopus stolonifer was strongly inhibited by the jelly fig chitinase as well. According to the above results, jelly fig chitinase has the thermostable and antifungal activity, and due to such kinds of traits, the jelly fig chitinase may be a potential fungicide in the future. Keywords : jelly fig, achene, chitinase, fungus, thermostable, pericarp.Table of Contents Chinese abstract…………………………………………………………………….1 English abstract…………………………………………………………………….3 Research background……………………………………………………………....6 References…………………………………………………………………………..8 Chapter 1 Ultrastructure of jelly fig (Ficus awkeotsang) achenes Abstract…………………………………………………………………………….10 Introduction………………………………………………………………………..11 Materials and Methods…………………………………………………………....12 Results……………………………………………………………………………...15 Discussion…..………………………………………………………………………19 References………………………………………………………………………….21 Figures……………………………………………………………………………...24 Chapter 2 Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes Abstract……………………………………………………………..………………37 Introduction…………………………………………………………..…………….38 Materials and Methods………………………………………………..…………...40 Results…………………………………………………………………..…………..46 Discussion…..……………………………………………………………..………...48 References…………………………………………………………………..………50 Tables………………………………………………………………………..………54 Figures………………………………………………………………………..……..57 Chapter 3 Antifungal activity of a chitinase in the pericarp of achenes Abstract…..………………………………………………………………………….67 Introduction…..……………………………………………………………………..68 Materials and Methods…..………………………………………………………....71 Results……………………..………………………………………………………...74 Discussion…………………...……………………………….………………………76 References……………………..…………………………………………………….78 Figures…………………………..…………………………………………………...83 Conclusion……………………..………………………………………..9

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【中文摘要】 蛋白二硫键异构酶(PDI)是真核生物重要的多功能蛋白,其基因结构在虾蟹类尚未报道.从拟穴青蟹Scylla pa-ramamosain(Estampador,1949)脑组织中分离纯化总RNA,经逆转录得到cDNA第一条链,并以之为模板利用RACE(rapid-amplification of cDNA ends)技术,扩增出一条950 bp的cDNA.将所得cDNA克隆到质粒载体pTZ57R/T,转化大肠杆菌DH5α细胞并对筛选的阳性克隆测序.通过与目前数据库中序列的比较,在此cDNA编码蛋白中发现其中101个氨基酸与其他物种已知蛋白二硫键异构酶(PDI)中的PDI_a_PDI_a′_C保守区域相似率在64%~79%之间.确定此cDNA编码拟穴青蟹PDI_a_PDI_a′_C保守区域. 【英文摘要】 The protein disulfide isomerase severs to be a multifunctional protein of great importance to the eukaryotes.The total RNAs isolated from the brain of Scylla paramamosainn(Estampador,1949) were reverse transcribed to the first strands of cDNA.A cDNA fragment of 950 bp was amplified from the first strands of cDNA by RACE(rapid-amplification of cDNA ends) method.This fragment was inserted into plasmid pTZ57R/T for screening and DNA sequencing.Compared with current sequences in the database,a 101-aa domain,whi...国家自然科学基金(40406030,40776084);福建省高等学校新世纪优秀人才支持计划资

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EFFECT OF Cu, Cd AND Cu:Cd MIXTURE ON THE BIOCHEMICAL COMPOSITION OF TWO PENAEID SHRIMP POSTLARVAE

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