Schwann Cell Migration Induced by Earthworm Extract via Activation of PAs and MMP2/9 Mediated through ERK1/2 and p38

The earthworm, which has stasis removal and wound-healing functions, is a widely used Chinese herbal medicine in China. Schwann cell migration is critical for the regeneration of injured nerves. Schwann cells provide an essentially supportive activity for neuron regeneration. However, the molecular migration mechanisms induced by earthworms in Schwann cells remain unclear. Here, we investigate the roles of MAPK (ERK1/2, JNK and p38) pathways for earthworm-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production in Schwann cells. Moreover, earthworm induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream signaling expression of PAs and MMPs in a time-dependent manner. Earthworm-stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with U0126 and SB203580, resulting in migration and uPA-related signal pathway inhibition. The results were confirmed using small interfering ERK1/2 and p38 RNA. These results demonstrated that earthworms can stimulate Schwann cell migration and up-regulate PAs and MMP2/9 expression mediated through the MAPK pathways, ERK1/2 and p38. Taken together, our data suggests the MAPKs (ERK1/2, p38)-, PAs (uPA, tPA)-, MMP (MMP2, MMP9) signaling pathway of Schwann cells regulated by earthworms might play a major role in Schwann cell migration and nerve regeneration

Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors

Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination

Structural and Functional Analyses of Five Conserved Positively Charged Residues in the L1 and N-Terminal DNA Binding Motifs of Archaeal RadA Protein

RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 Å pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target

The FRAP of Vegetables Consumed in Taiwan as Related to Total Phenolic Content

蔬果提供人體抗氧化的保健作用，多攝取可減少疾病的發生。為了建立台灣蔬菜的保健資訊，本試驗以三價鐵離子還原抗氧化力分析法（FRAP assay）縱觀分析台灣各種蔬菜的總抗氧化力，並利用福林酚試劑（Folin-Ciocalteu reagent）分析法量測其總酚類含量。所有受測蔬菜的平均抗氧化力介於0.4~35.7μmol FeSO4/g f.w.，總酚類含量介於0.7~47.7μmol GAE/g f.w. 高總抗氧化力（>10μmol FeSO4/g f.w.）的蔬菜有短錐形紅辣椒、薑、甜椒、紫色甘藍、黑皮長豇豆、甘藷葉、羅勒（九層塔）、蓮藕、豌豆嫩梢及過溝菜蕨（過貓），這些蔬菜同時也具高總酚類含量（>10μmol GAE/g f.w.）。蔬菜以供食用部位分類，茄果類平均抗氧化力9.6μmol FeSO4/g f.w.為最高，瓜果類1.6μmol FeSO4/g f.w.最低；總酚類含量方面則皆無顯著差異。濃綠色以及顏色鮮艷的蔬菜，抗氧化力與總酚類含量皆較高於白色或淡色的蔬菜。將受試蔬菜依中醫熱、溫、涼及寒四氣性質分類，結果指出各類蔬菜平均抗氧化力與總酚類含量皆呈現由高到低的順序。本試驗受測78種蔬菜的單位鮮重的抗氧化力與總酚類含量有正相關（R2=0.46，p&lt;0.001）；其中，豆科、瓜果類及茄科等蔬菜抗氧化力與總酚類含量有更高度的相關性，決定係數（R2）分別為0.91、0.88及0.80，而十字花科與菊科蔬菜的決定係數則不高（R2分別為0.23及0.28），結果顯示不同類別的蔬菜，總酚類對總抗氧化力的貢獻度不同，抗氧化成分有所差異。在不同部位與不同前處理的蔬菜抗氧化力方面，小松菜與本地芹菜葉片之葉身部位抗氧化力是高於葉柄部位；蓮藕地下莖之節位部份的單位乾重抗氧化力是節間部位的3.5倍；大蒜經切碎後放置20分鐘再進行均質分析，其抗氧化力可提高50%；而含有外皮的絲瓜單位乾重抗氧化力是高於去皮的絲瓜。預期外地，包心白菜單位乾重抗氧化力，有由內層葉片到外層葉片遞減的趨勢。由國人蔬菜攝取的情況，可估計出台灣成人平均每天從蔬菜類食物中所獲得的抗氧化力為1442μmol FeSO4，所獲得的總酚類為1524μmol GAE，其中深色蔬菜類所提供的抗氧化力與總酚類皆約佔全部的75%。Fruit and vegetables play an important role in antioxidant protective effect for human against the risk of several diseases. To establish information of health-promoting properties of Taiwan vegetables, the total antioxidant activity（TAA）and total phenolic content（TP）of Taiwan vegetables were investigated by Ferric reducing antioxidant power（FRAP）assay and Folin-Ciocalteau assay. The TAA and TP varied from 0.4 to 35.7μmol FeSO4/g f.w. and from 0.7 to 47.7μmol GAE/g f.w., respectively. The vegetables with higher TAA (&gt;10μmol FeSO4/g f.w.) and TP (&gt;10μmol GAE/g f.w.) are short chili pepper, ginger, colored sweet pepper, red cabbage, black yard-long bean, sweet potato vine, basil, Indian lotus root, early dwarf pea, and common bracken. The TAA was the highest in fruits of Solanaceae and the lowest in fruits of Cucurbitaceae. The TAA and TP were significantly higher in colorful vegetables than in light color vegetables. As classified the tested vegetables according to hot, warm, cool, and cold properties of Chinese medicine, the average TAA and TP of each category were in the decrement order. A positive linear relationship (R2 =0.46, p<0.001) obtained between TAA and TP in 78 kinds vegetables. The coefficient of determination between TAA and TP was higher in Leguminous, Cucurbitaceous and Solanaceous vegetables (r2=0.91, 0.88, 0.80, respectively), and lower in Brassicaceous and Asteraceous vegetables (r2=0.23 and 0.28, respectively). These results suggested that the contribution of TP to TAA varies among varieties of vegetable, and antioxidants as well. The TAA of Komatsuna and Chinese celery were higher in the leaf blade than in the leaf stalk, and the TAA of Indian lotus root was 3.5 fold higher in nodes than in internodes. he TAA of garlic in creased 50% as left at room temperature for 20 minutes after minced before extraction. The TAA of vegetable sponge was higher in fruit with skin than in ones without skin. Unexpectedly, Chinese cabbage has the decrement in TAA from inner to outer leaves. The average TAA and TP consumed from vegetables are 1442μmol FeSO4 and 1524μmol GAE per adult in Taiwan daily. The percentage of TAA and TP consumed from deep-colored vegetables is about 75% in all vegetables.目 錄試委員會審定書 謝…………………………………………………………………………………… i文摘要……………………………………………………………………………… ii文摘要……………………………………………………………………………… iii一章 前言………………………………………………………………………… 1二章 前人研究………………………………………………………………… 3、 蔬果抗氧化的保健功能……………………………………………………3、 蔬果對人體抗氧化的相關研究……………………………………………4、 蔬果的抗氧化物質與其抗氧化作用………………………………………6、 抗氧化力分析方法…………………………………………………………10、 蔬果抗氧化力的相關研究…………………………………………………13、 國人蔬菜攝取情況與國內蔬菜供給情況…………………………………15三章 台灣蔬菜鐵離子還原抗氧化力（FRAP）之研究…………………………24、前言…………………………………………………………………………24、材料與方法…………………………………………………………………27、結果與討論……………………………………………………………… 32一） FRAP分析方法的建立…………………………………………………32二） 台灣蔬菜FRAP的研究…………………………………………………41三） 不同部位與經過不同前處理的蔬菜FRAP……………………………52四） 蔬菜攝取情況與獲取抗氧化力的關係…………………………………58五） 國內蔬菜供給量與國人獲取蔬菜抗氧化力的關係……………………60四章 台灣蔬菜總酚類含量之研究………………………………………………62、前言…………………………………………………………………………62、材料與方法…………………………………………………………………64、結果與討論……………………………………………………………… 67一） 總酚類含量分析方法的建立……………………………………………67二） 台灣蔬菜總酚類含量的研究……………………………………………73三） 蔬菜攝取情況與獲取總酚類含量的關係………………………………84四） 國內蔬菜供給量與國人獲取蔬菜總酚類的關係………………………85五章 結論………………………………………………………………………88考文獻………………………………………………………………………………89錄…………………………………………………………………………………10