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Tu ya ji
No full textV.1. 塗鴉集 : 卷上 -- v.2. 塗鴉集 : 卷下 -- v.3. 塗鴉集雜錄 -- 塗鴉集 : 文部贊 -- 塗鴉集 : 文部啟.V.1. Tu ya ji : juan shang -- v.2. Tu ya ji : juan xia -- v.3. Tu ya ji za lu -- Tu ya ji : wen bu zan -- Tu ya ji : wen bu qi.一機.綫裝, 1函.框20.3x13.5公分, 9行20字. 白口, 四周雙邊, 單黑魚尾. 版心上鐫題名, 中鐫卷次, 下鐫葉次.分上, 下卷.出書年據序.Xian zhuang, 1 han.Kuang 20.3 x 13.5 gong fen, 9 hang 20 zi. Bai kou, si zhou shuang bian, dan hei yu wei. Ban xin shang juan ti ming, zhong juan juan ci, xia juan ye ci.Fen shang, xia juan.Chu shu nian ju xu.Yiji
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Wave Propagation in Flexible Tubes Immersed in Fluid
No full text為探究超音波對血管的影響，本文研究置於流體中的柔性管與流體之間交互作用的頻散現象與波傳模態。 根據Chebyshev-Gauss-Lobatto插值法與微分矩陣原理，採用Spectral數值方法離散彈性力學理論的波動方程，建立軸對稱縱向、非軸對稱周向和彎曲模態對應的廣義特徵值問題。此法克服Bessel函數的發散問題，成功解出頻譜、模態中各物理量的振形與質點軌跡圖。 第一、第二頻散曲線不論在低頻或高頻區域皆對應於彈性管表面波模態，分別為反對稱與對稱模態；第三、第四頻散曲線在兩相速度尚未收斂至相當接近的區域時，皆為管內外流體表面波模態，且在管面上流體振幅比管振幅大，沿兩頻散曲線移動，隨頻率增加兩相速度值逐漸靠近，第三頻散曲線最終收斂至管外流體表面波模態，第四頻散曲線收斂至管內流體表面波模態。 對應於EKOS的2MHz高頻模式，能量會集中在血管表面，血管內部振幅極小；而操作在45kHz的低頻模式時，血管內部會承受較大的振幅。因此以2MHz高頻模式操作對血管的傷害較低。The wave propagation in flexible tubes immersed in fluid is studied in order to explore the effect of ultrasonic waves incident on blood vessels. Based on Chebyshev-Gauss-Lobatto interpolation and differential matrix, spectral method discretizes the Helmholtz equation to establish generalized eigenvalue problems. These generalized eigenvalue problems are formulated for axial symmetric, circumferential, and bending modes respectively. The numerical method overcomes the divergent problem of Bessel functions and the spectrum, mode shapes, and motion of particles are found successfully. Mode shapes corresponding to the first and second dispersion curves display surface wave patterns of the tube in a very wide frequency range. The first dispersion curve gives antisymmetric modes while the second one offers symmetric modes. Before convergence, the third and fourth dispersion curves provide interface waves of fluid on both inside and outside of tubes. As frequency is increased, mode shapes corresponding to the third dispersion curve converge to surface waves of fluid outside the tube and that corresponding to the fourth dispersion curve converge to surface waves of fluid inside the tube. For EKOS operated at high frequency mode of 2MHz, surface wave modes will be excited most due to the ultrasonic source location. Therefore, the ultrasonic energy is mainly concentrated in the vicinity of vessel surface. When it is operated at low frequency mode of 45kHz, there is more ultrasonic energy penetrating into the vessel wall than that of 2MHz mode. That is, high frequency operation mode causes less damage to the vessel than low frequency operation mode
Photodynamic Inactivation against Bacterial Planktonic Cells and Biofilms with C-Phycocyanin Extracted from Spirulina platensis
No full text光動力治療(Photodynamic therapy, PDT) 是一種使用特定波長
之激發光將光感物質(photosensitizer) 激發成高能量激發態，經由能
量或電子傳遞，將細胞周圍氧分子轉變成有細胞毒性的單態氧
(1O2)，或與環境分子作用產生自由基，造成細胞死亡的方法，目前已
應用於癌症治療及微生物防治(Photodynamic inactivation, PDI)。研究
顯示，將光動力治療應用在微生物防治上已有部分成效，而積極開發
新光源及光感物質，使光動力治療能更有效發揮，為目前研究的新趨
勢。藻藍素(phycocyanin, PC) 為螺旋藻(Spirulina platensis) 中的一
種水溶性螢光蛋白，可吸收光能並進行能量及電子傳遞，具有光動力
治療之潛力。本實驗由螺旋藻Spirulina platensis 中萃取藻藍素作為光
感物質，並以發光二極體(Light emitting diodes，LED) 為光源，對
革蘭氏陽性及陰性菌進行光動力抑制，並進一步探討菌體在懸浮狀態
與生物膜狀態下，光動力殺菌效果是否不同。實驗結果顯示由螺旋藻
中萃取並純化出藻藍素，回收率可達56.1%，純度(A620nm/280nm) 為
4.56，其品質與商品化之藻藍素相當。而以藻藍素進行光動力治療，
在藻藍素濃度100 µg ml-1，照光強度360 J cm-2 之條件下，可將濃度
107 CFU ml-1 之革蘭氏陽性菌S. aureus 及S. epidermis 懸浮細胞完全
抑制，但對革蘭氏陰性菌E. coli 及P. aeruginosa 之效果則不顯著；
而以藻藍素對S. aureus 及S. epidermis 生物膜進行光動力抑制，在藻
藍素濃度900 µg ml-1，照光強度360 J cm-2 之條件下。亦可將108 CFU
cm-2 之生物膜菌體完全殺滅。此研究顯示由螺旋藻中萃取之天然光感
II
物質藻藍素對革蘭氏陽性菌之懸浮菌體及生物膜細胞皆有良好之光
動力抑制效果，在未來的應用上極具潛力。
關鍵詞：光動力抑制、螺旋藻、藻藍素、生物膜Photodynamic inactivation (PDI) utilized photosensitizers and light
with appropriate wave length to give a phototoxic response, normally via
oxidative damage. Although PDI might be an effective approach in
antimicrobial treatment, no photosensitizer is suitable for all possible
applications. Therefore, the development of new photosensitizers became
important to overcome the shortage of PDI. Phycocyanin (PC), a
water-soluble non-toxic biliprotein, is one of the major constituents of
Spirulina platensis. The photobiological properties of PC suggested its
possibility to be a photosensitizer in photodynamic therapy. The goal of
this study was to investigate the effect of the PC extracted from Sp.
platensis on photodynamic inactivation against bacterial planktonic and
biofilm cells. The extraction and purification of PC was accomplished by
fractional precipitation with ammonium sulfate precipitation, following
by ion-exchange chromagraphy on Macro-Prep DEAE Support system.
The purity of PC was examined by absorbance and fluorescence
spectrometry. Both Gram-positive and Gram-negative bacteria were
tested for PC-PDI. Pure PC was finally obtained from Sp. platensis with
purity ratio (A620nm/280nm) 4.56 and recovery rate 56.1%. After PC-PDI
treatment, which carried with 100 µg ml-1 of PC and irradiation light dose
of 360 J cm-2, no viable cell of the Gram-positive Staphylococcus aureus
and S. epidermis planktonic cells were detected. However, the
Gram-negative Escherichia coli and Pseudomonas aeruginosa were
resistant to the PC-PDI treatment. Experiments with PC-PDI against S.
aureus and S. epidermis biofilms showed that both species of the tested
biofilms were sensitive to the PC-PDI treatment, with a decrease of about
8-log of viable cells. This study showed that the PC from Sp. platensis
was a potential photosensitizer to inactivated Gram-positive bacteria
planktonic cells and biofilms.
Keyword: Photodynamic Inactivation, Spirulina platensis, Phycocyanin,
Biofilm目錄
中文摘要------------------------------------------------------------------------- I
英文摘要----------------------------------------------------------------------- III
目錄------------------------------------------------------------------------------- i
表次------------------------------------------------------------------------------- v
圖次------------------------------------------------------------------------------ vi
壹、前言-------------------------------------------------------------------------- 1
1. 生物膜------------------------------------------------------------------------ 1
1.1 環境中之生物膜------------------------------------------------------ 1
1.2 生物膜之形成--------------------------------------------------------- 2
1.3 生物膜之結構--------------------------------------------------------- 5
1.4 生物膜之抗藥性機制------------------------------------------------ 7
1.4.1 物理屏障-------------------------------------------------------- 7
1.4.2 生理適應-------------------------------------------------------- 7
1.4.3 細胞對細胞的聯繫-------------------------------------------- 7
1.5 生物膜防治------------------------------------------------------------ 8
2. 光動力抑制------------------------------------------------------------------ 9
2.1 光動力作用之歷史與應用------------------------------------------ 9
2.2 光動力作用之機制------------------------------------------------- 10
2.3 光動力作用之要素------------------------------------------------- 15
2.3.1 光源------------------------------------------------------------ 15
2.3.2 光感物質------------------------------------------------------ 17
2.3.3 氧氣------------------------------------------------------------ 18
ii
2.4 光感物質之開發---------------------------------------------------- 19
2.5 天然光感物質------------------------------------------------------- 21
3. 藻藍素---------------------------------------------------------------------- 22
3.1 螺旋藻之經濟價值------------------------------------------------- 22
3.2 藻藍素在生物體之角色------------------------------------------- 23
3.3 藻藍素之醫療價值------------------------------------------------- 26
3.4 藻藍素之光感性質及潛力---------------------------------------- 29
3.5 萃取方法之研究回顧---------------------------------------------- 31
4. 動機與目的---------------------------------------------------------------- 32
貳、材料與方法---------------------------------------------------------------- 34
1 菌種------------------------------------------------------------------------- 34
2 儀器------------------------------------------------------------------------- 34
2.1 藻藍素純化---------------------------------------------------------- 34
2.2 生物膜培養系統---------------------------------------------------- 34
2.3 光動力作用裝置---------------------------------------------------- 36
3 實驗材料與方法---------------------------------------------------------- 37
3.1 緩衝溶液之配製---------------------------------------------------- 37
3.1.1 磷酸鉀緩衝溶液之配製------------------------------------ 37
3.1.2 等張磷酸鹽緩衝溶液之配製------------------------------ 37
3.2 懸浮菌體培養與定量---------------------------------------------- 37
3.2.1 金黃色葡萄球菌之培養------------------------------------ 37
3.2.2 白色表皮葡萄球菌之培養--------------------------------- 38
3.2.3 大腸桿菌之培養--------------------------------------------- 38
3.2.4 綠膿桿菌之培養--------------------------------------------- 38
iii
3.2.5 活菌數定量方法--------------------------------------------- 38
3.3 生物膜培養與定量------------------------------------------------- 39
3.3.1 S. aureus 與S. epidermis 生物膜之培養------------------ 39
3.3.2 生物膜之定量------------------------------------------------ 39
4 藻藍素純化與分析------------------------------------------------------- 39
4.1 藻藍素粗萃取------------------------------------------------------- 39
4.2 藻藍素純化---------------------------------------------------------- 40
4.3 藻藍素定性分析---------------------------------------------------- 40
4.3.1 聚丙烯醯胺膠體電泳--------------------------------------- 40
4.3.2 液相層析質譜儀分子量決定法--------------------------- 40
4.3.3 測定藻藍素之光譜特性分析------------------------------ 40
4.4 藻藍素定量分析---------------------------------------------------- 41
4.4.1 蛋白質定量法------------------------------------------------ 41
4.4.2 藻藍素吸收光譜分析--------------------------------------- 41
4.4.3 藻藍素放射光譜分析--------------------------------------- 41
5 藻藍素之光動力抑制實驗---------------------------------------------- 42
5.1 藻藍素對懸浮菌體之光動力抑制------------------------------- 42
5.2 藻藍素對生物膜之光動力抑制---------------------------------- 42
参、結果與討論---------------------------------------------------------------- 43
1. 生物膜培養與定量------------------------------------------------------- 43
1.1 金黃色葡萄球菌生物膜之生長曲線---------------------------- 43
1.2 白色表皮葡萄球菌生物膜之生長曲線------------------------- 45
2. 藻藍素萃取與純化------------------------------------------------------- 46
2.1 萃取與純化條件---------------------------------------------------- 46
iv
2.1.1 硫酸銨沈澱法濃度條件測試與結果--------------------- 46
2.1.2 Rivanol 處理法之比較-------------------------------------- 47
2.1.3 藻藍素純化結果--------------------------------------------- 49
2.2 藻藍素定性分析---------------------------------------------------- 50
2.2.1 藻藍素分子量------------------------------------------------ 50
2.2.2 藻藍素之吸收光譜------------------------------------------ 52
2.2.3 藻藍素之放射光譜------------------------------------------ 53
2.3 藻藍素定量分析---------------------------------------------------- 55
3. 藻藍素之光動力抑制實驗---------------------------------------------- 58
3.1 藻藍素對懸浮菌體之光動力抑制作用------------------------- 58
3.1.1 革蘭氏陽性菌------------------------------------------------ 58
3.1.2 革蘭氏陰性菌------------------------------------------------ 64
3.2 藻藍素對生物膜之光動力抑制作用---------------------------- 67
3.2.1 金黃色葡萄球菌生物膜------------------------------------ 67
3.2.2 白色表皮葡萄球菌生物膜--------------------------------- 69
4. 討論------------------------------------------------------------------------- 70
4.1 藻藍素萃取與純化------------------------------------------------- 70
4.2 藻藍素之光動力抑制效果---------------------------------------- 71
4.3 藻藍素之光動力作用對革蘭氏陽性菌與陰性菌之差別---- 74
4.4 藻藍素之光動力作用對懸浮細胞與生物膜之抑制效果---- 77
肆、結論與未來展望---------------------------------------------------------- 78
伍、參考文獻------------------------------------------------------------------- 79
表次
表一、藻藍素基本性質------------------------------------------------------- 30
表二、液相層析質譜儀分析結果------------------------------------------- 51
表三、藻藍素萃取與純化結果---------------------------------------------- 57
表四、純化結果與前人研究之比較---------------------------------------- 70
表五、藻藍素之光動力抑制結果與前人研究之比較------------------- 73
圖次
圖一、生物膜之形成示意圖-------------------------------------------------- 4
圖二、生物膜之蕈狀結構假想圖-------------------------------------------- 7
圖三、光動力作用機制------------------------------------------------------- 12
圖四、光動力作用之能量轉移---------------------------------------------- 13
圖五、光感物質與環境分子間之作用------------------------------------- 14
圖六、藻膽體構造------------------------------------------------------------- 24
圖七、不同色素之吸收光譜------------------------------------------------- 25
圖八、藻藍素之發色團------------------------------------------------------- 27
圖九、藻藍素結構------------------------------------------------------------- 28
圖十、生物膜反應器及裝置------------------------------------------------- 35
圖十一、紅色發光二極體---------------------------------------------------- 36
圖十二、金黃色葡萄球菌生物膜之生長曲線---------------------------- 44
圖十三、白色表皮葡萄球菌生物膜之生長曲線------------------------- 45
圖十四、藻藍素粗萃取液經不同濃度之硫酸銨沈澱結果------------- 47
圖十五、兩種不同萃取方式------------------------------------------------- 48
圖十六、純化所得之藻藍素------------------------------------------------- 49
圖十七、13% 聚丙烯醯胺膠體電泳結果--------------------------------- 51
圖十八、藻藍素吸收光譜---------------------------------------------------- 52
圖十九、藻藍素放射光譜---------------------------------------------------- 53
圖二十、不同濃度藻藍素之放射光譜------------------------------------- 54
圖二十一、濃度與光譜強度之線性關係---------------------------------- 56
圖二十二、以藻藍素標準品對革蘭氏陽性菌S. aureus 懸浮細胞進行
vii
光動力抑制----------------------------------------------------- 60
圖二十三、以藻藍素標準品對革蘭氏陽性菌S. epidermis 懸浮細胞進
行光動力抑制-------------------------------------------------- 61
圖二十四、以藻藍素樣品對革蘭氏陽性菌S. aureus 懸浮細胞進行光
動力抑制-------------------------------------------------------- 62
圖二十五、以藻藍素樣品對革蘭氏陽性菌S. epidermis 懸浮細胞進行
光動力抑制----------------------------------------------------- 63
圖二十六、以藻藍素標準品對革蘭氏陰性菌P. aeruginosa 懸浮細胞進
行光動力抑制-------------------------------------------------- 65
圖二十七、以藻藍素標準品對革蘭氏陰性菌E. coli 懸浮細胞進行光動
力抑制----------------------------------------------------------- 66
圖二十八、以藻藍素樣品對S. aureus 生物膜進行光動力抑制------- 68
圖二十九、以藻藍素樣品對S. epidermis 生物膜進行光動力抑制--- 69
圖三十、藻藍素激發量與菌株之關係------------------------------------- 75
圖三十一、菌體周圍之藻藍素濃度---------------------------------------- 7
The Gating Mechanism of the NMDA Receptor Channel
No full textNMDA離子通道分別是由兩個GluN1和兩個GluN2所組成，然而這兩個不同的次單元在通道上的貢獻還不是很清楚。我們發現glycine對GluN1的親和力(K gly ∼ 0.6 μM)明顯高於NMDA或glutamate對GluN2的親和力(K NMDA ∼ 36 μM, K glu ∼ 4.8 μM)。然而Glycine對GluN1的結合速度(∼9.8 × 106 M-1 s-1) 稍為快於NMDA 對 GluN2 (∼4.1 × 106 M-1 s-1)。Glycine從GluN1離開結合位的速度(time constant ∼2 s)明顯慢於NMDA從GluN2離開結合位的速度(time constant ∼70 ms)。除此之外，同時洗掉glycine和NMDA的NMDA通道decay速度和NMDA離開結合位的速度相似。但是如果只洗掉glycine，NMDA通道decay速度明顯變慢，且和glycine離開結合位相似。在缺乏給予NMDA的情況下，glycine的結合速度對於離開結合位的通道，大概和共同給予NMDA和glycine相似。然而在缺乏給予glycine下的NMDA結合速度明顯變慢。另外，NMDA離子通道的GluN1和GluN2上有一段保留序列SYTANLAAF，其第七個胺基酸A7在GluN1和GluN2上非常靠近可以控制外口的大小。除此之外，突變在GluN1的A7可以改變glycine和NMDA對離子通道的親和力，但在GluN2則只有影響NMDA對通道的親和力。因此，GluN2是負責活化的門閥，而GluN1扮演調控門閥的角色。結構上，調控次單元似乎是透過A7的胺基酸。 除此之外，NMDA對於鈣離子有相當高的通透性，而鈣離子的通透會影響許多細胞生物的性質。NMDA具有鈣離子依賴性的去敏感性，而目前研究顯示鈣離子和胞內的calcineurin等物質會交互作用產生鈣離子依賴性的去敏感性，然而由於在外口也有鈣離子的結合位，鈣離子是否也會在外口作用，產生鈣離子依賴性的去敏感性。我們發現在外口的鈣離子和鉻離子（和鈣離子有相同價電子數和半徑）會結合到休息態的NMDA而影響門閥的開關。由於活化門閥位於外口處，這些離子的結合位應該處於更外口的地方。根據我們結果顯示鉻離子鉻離子對通道是一對一的結合，其對休息態,活化態,去敏感態的親和力分別為分別為~5, ~2.5, and ~1.2 μM。DRPEER motif(在GluN1而不在GluN2)位於活化門閥的外面，當DEE突變成Alanine時則Cd離子的親和力無論是休息態或是活化態會下降，因此鈣或鉻離子可能透過DRPEER motif進而影響活化門閥，鈣或鉻離子的抑制能力在T647A突變也會下降，且會產生明顯的hook電流，暗示這突變的通道只有休息態和活化態，沒有去敏感態，也暗示去敏感態和conformational的改變有關。 另一方面，去敏感態在NMDA扮演重要的角色，目前切確的調控機轉仍然未知，我們發現tetrapentylammonium (TPentA)接在外口會有gating modify的角色，使通道喜歡待在開啟的裝態，而不喜歡待在去敏感態。另外SYTANLAAF在gating上也扮演很重要的角色，像是A7或是T3突變之後對desensitization都有嚴重的影響，暗示desensitization是一種結構上的改變，而且可能就在活化閘門A7的附近。The NMDA receptor channel is an obligatory heterotetramer formed by two GluN1 and two GluN2 subunits. However, the differential contribution of the two different subunits to channel operation is not clear. We found that the apparent affinity of glycine to GluN1 (Kgly~0.6 μM) is much higher than NMDA or glutamate to GluN2 (KNMDA~36 μM, Kglu~4.8 μM). The binding rate constant (derived from the linear regression of the apparent macroscopic binding rates) of glycine to GluN1 (~9.8 x 106 M-1s-1), however, is only slightly faster than NMDA to GluN2 (~4.1 x 106 M-1s-1). Accordingly, the apparent unbinding rates of glycine from activated GluN1 (time constant ~2s) are much slower than NMDA from activated GluN2 (time constant ~70 ms). Moreover, the decay of NMDA currents upon wash-off of both glycine and NMDA seems to follow the course of NMDA rather than glycine unbinding. But if only glycine is washed off, the current decay is much slower, apparently following the course of glycine unbinding. The apparent binding rate of glycine to the fully deactivated channel, in the absence of NMDA, is roughly the same as that measured with co-application of both ligands, whereas the apparent binding rate of NMDA to the fully deactivated channel in the absence of glycine is markedly slower. In this regard, it is interesting that the 7th residue in the highly conserved SYTANLAAF motif (A7) in GluN1 and GluN2 are so close that they may interact with each other to control the dimension of the external pore mouth. Moreover, specific mutations involving A7 in GluN1 but not in GluN2 result in channels showing markedly enhanced affinity to both glycine and NMDA and readily activated by only NMDA, as if the channel is already partially activated. We conclude that GluN2 is most likely directly responsible for the activation gate of the NMDA channel, whereas GluN1 assumes a role of more global control, especially on the gating conformational changes in GluN2. Structurally, this inter-subunit regulatory interaction seems to involve the SYTANLAAF motif, especially the A7 residue. Furthermore, the NMDA receptor channel is characterized by its high permeability of Ca2+ ion, and the Ca2+ influxes may play an important role in many cellular physiological and pathophysiological processes. It has been reported that NMDA channel desensitization, an imperative attribute regulating ionic fluxes through the channel pore, could be related to Ca2+ ions flowing through the pore and/or interacting with effector proteins such as calcineurin at the internal pore mouth. Whether extracellular Ca2+ ion itself could directly bind to the NMDA receptor channel to have an effect on the key molecular behaviors of the channel has not been fully characterized. We found that extracellular Ca2+ and Cd2+, an ion with the same charges and ionic radius as Ca2+ and therefore high affinity toward many Ca2+ binding sites, could bind to the closed NMDA channel to affect channel gating as well as ion permeation. Because the activation gate, which is already positioned at the external pore mouth, is not open, this binding site must be located at the very external part of the pore. We also demonstrated that Cd2+ binds to the NMDA channel with a simple bimolecular reaction, and the apparent dissociation constants toward the closed, open, and desensitized states of the channel is ~5, ~2.5, and ~1.2 μM, respectively. The modest but definite differential affinity would indicate that the binding site changes its conformation during the gating process. DRPEER, a motif in the GluN1 but not GluN2 subunit, is located just external to the activation gate of the NMDA channel. Interestingly, the effect of Cd2+ present in either the resting or the activated state is decreased correlatively to the number of charge-neutralization mutations in this motif, with additive free energy changes calculated by double mutant cycle analysis. DRPEER motif therefore very likely constitutes the binding site for Ca2+ or Cd2+, and thus seems to go through a sequential conformational change during channel activation. In this regard, it is intriguing that the inhibitory effect of Cd2+ is also decreased by point mutation T647A located just inside the activation gate in the pore. Moreover, prominent “hooked” current develops after wash-off of Cd2+ (and even more so after wash-off of Ca2+) in this case, suggesting preservation of the channel in the open state (prevention of the channel from entering the desensitized state) and thus an apparently inversed order of affinity of Cd2+ and Ca2+ toward the two states with the point mutation. Desensitization thus seems to involve essential conformational changes in the vicinity of the activation gate in the NMDA channel. Desensitization is an important gating mechanism for the function of N-methyl-D-aspartate (NMDA) receptors. However, the exact modulation of desensitization mechanism remains in question. We found that extracellular tetrapentylammonium (TPentA) binds to the external pore mouth, and induces a strong increase in open probability to disturb the equivalence between open and desensitization states. TpentA seems to prefer to open state of the NMDA channel. Moreover, the A7 residues (at position 7 of the SYTANLAAF motif), shown to mark the activation gate, constitute the key component of the activation gate of the NMDA channel. We further explore whether A7 is also involved in the desensitization. Our data suggest that the desensitization gate should be near the activation gate, A7, in the external pore mouth
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
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