Oxidative Stress Disruption of Receptor-Mediated Calcium Signaling Mechanisms

Background: Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [3H]N-methylscopolamine ([3H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca2+]i and [Ca2+]L, respectively) were measured by fluorescent imaging. Results: Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP. Conclusions: Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling

十字花科黑腐病菌致病相關基因之分離與定性

Five Xanthomonas campestris pv. campestris (Xcc) isolates with different virulence capability were used in this study in order to isolate virulence genes of the bacteria. . Xcc ATCC33913, Xcc59, Xcc17 are high-virulence isolates, Xcc11 is a low-virulence isolate and Xcc11A is a spontaneous avirulent mutant of Xcc11. Of the 5 isolates, the whole genome sequence of Xcc ATCC33913 is known. Southern hybridization analysis using multi-copy IS1478 as probe revealed very similar hybridization patterns between Xcc17 and Xcc11, and Xcc11 and Xcc11A. Two DNA fragments (7E23, 7P23) from Xcc17 corresponding to the different hybridization signals between Xcc17 and Xcc11, and one DNA fragment (1E43) from Xcc11 corresponding to the different hybridization signal between Xcc17 and Xcc11 were cloned and sequenced. The sequence results showed that 7P23 and 1E43 were probably not related to pathogenicity of Xcc17 Xcc11 and Xcc11A. 7E23, however, contains a 308-bp DNA sequence between two copies of IS1478, which is identical to the 3' end, and the downstream sequence of the truncated NTPase gene in Xcc ATCC33913. The protein sequence of the truncated NTPase gene showed 45% homology to the sequence of VirD4, which is a component of the type IV secretion pathway in many bacteria involved in secretion of virulence factors. PCR studies showed that Xcc17, Xcc11, and Xcc11A contain the virD4 gene. By using virD4 gene as probe, southern hybridization analysis showed that Xcc11A lacked a 3.0 kb HindIII hybridization fragment, as compared to the hybridization pattern of Xcc11. Additionally, both Xcc11 and Xcc11A carry an extra 1.5 kb fragment between virD4 and virB8, another component of the type IV secretion pathway. These DNA fragments were cloned and sequenced. An Xcc 17 mutant that has transcriptional terminator inserted in the region between virD4 and virB8 was constructed and its pathogenecity was remained the same. On the other hand, the 3.0 HindIII fragment of Xcc11 contains an orf which was named vgA1. The VgA1 sequence showed 45% homology to the VirD4 sequence. The vgA1 knock-out mutant of Xcc11 lost the pathogenicity. The vgA1 is a pathogenicity gene of Xcc11 and might have role other than as a component of the type IV secretion pathway.本實驗使用五株具有不同致病能力的 Xanthomonas campestris pv. campestris (Xcc)，其中 Xcc ATCC33913、Xcc59，及 Xcc17為高致病力菌株，Xcc11 為低致病力菌株，而 Xcc11A 為 Xcc11產生自發性突變的無致病力菌株。另外 Xcc ATCC33193 之染色體序列以完全被定序發表。本實驗的目的是希望利用這些 Xcc 菌株找出 Xcc 致病性相關基因並進行分析。以轉位子 IS1478 進行南方雜配分析，發現 Xcc17 與 Xcc11的 hybridization pattern 極為相似，並選殖二個在 Xcc17 較 Xcc11 多出的雜配訊號 之 DNA片段 (7P23 及 7E23)，以及選殖一個在 Xcc11 較 Xcc17 多出的雜配訊號 之 DNA片段 (1E43)。定序比對後發現差異片段序列 7P23 與 1E43 可能與 Xcc 的致病力無關，而 7E23 片段包含二套反向的 部分IS1478，並且在此二套 IS1478 之間的序列為 truncated NTPase 基因 3’端的區域。Truncated NTPase 之氨機酸序列與 Xcc ATCC33913 中，屬於第四型分泌系統成員的 VirD4 蛋白序列有 45% 的相同性。 PCR 結果顯示 Xcc59、Xcc17、Xcc11，及 Xcc11A 同樣也含有virD4 基因。使用 virD4當探針進行南方雜配分析時，發現 Xcc11A 缺少一個 3.0 kb 的雜配訊號，並帶有一 virD4 的同質基因 vgA1。而在 Xcc11 及 Xcc11A 的第四型分泌系統基因群中，在 virD4 及 virB8 之間多出一 1.5 kb 的片段。選殖定序後發現 Xcc11 及 Xcc11A 在第四型分泌系統基因群中有一 IS1478 的插入。將一轉譯終止序列插入 Xcc17 相同的位置，發現此突變株的致病能力與 Xcc17 一樣。然而將 Xcc11 的vgA1 基因破壞後會失去致病能力。推測 vgA1基因為 Xcc11的致病基因，並且可能具有構成第四型分泌通道以外的功能。縮寫字對照表 4 中文摘要 6 英文摘要 7 前言 8 材料與培養條件 11 一、材料 11 二、培養條件 11 試劑與方法 12 一、各種培養基 12 二、少量細菌質體 DNA 的抽取 13 三、細菌染色體 DNA 的抽取 13 四、勝任細胞的製備 14 五、細菌轉型作用 15 六、三親交配 15 七、DNA 片段的回收 15 八、DNA 濃度測定 16 九、補平作用 17 十、DNA 黏接作用 17 十一、核酸定序 17 十二、南方雜配法 18 十三、引子的的合成 20 十四、聚合連鎖反應 (Polymerase Chain Reaction：PCR) 20 十五、十二烷基硫酸鈉聚丙烯醯氨板膠電泳法 SDS-PAGE 21 十六、西方雜配分析 23 十七、蛋白質濃度測定 24 十八、X. campestris pv. campestris 的胞外蛋白之誘導 25 十九、X. campestris pv. campestris 致病能力測試 26 研究結果 27 壹、Xcc 菌致病相關基因之選殖與定性 27 一、Xcc 之 Intergenic spacer (ITS) 序列分析 27 二、選殖 Xcc17 及 Xcc11，具有 IS1478 雜配訊號之差異片段 27 三、IS1478 雜配訊號之差異片段的定序與分析 28 貳、Xcc 菌第四型分泌系統與 Xcc 菌致病能力之探討 30 一、比較各 Xcc 菌之第四型分泌系統基因是否具有差異 30 二、經 HindIII 切割， Xcc11 位於 3 kb 較 Xcc11A 多出的virD4 雜配訊號之定性 32 三、Xcc17 第四型分泌系統表現抑制突變株的構築 32 四、Xcc11 之 vgA1::Kmr 突變株的構築 34 五、Xcc 突變株胞外蛋白分泌的能力 35 六、Xcc17GMW-6 突變株致病性實驗 35 七、Xcc11Km-9 突變株致病性實驗 36 八、測試 Xcc17GMW-6 突變株分泌OrfF’蛋白的能力 36 討論 37 參考文獻 40 表一、本實驗所是用之菌種 44 表二、本實驗所使用之質體 46 表三、本實驗所使用之引子 48 表四、Xcc 菌小苗戳莖實驗之結果 50 表五、Xcc 菌小苗剪葉實驗之結果 51 圖一、Xcc ATCC33913 第四型分泌系統的基因在染色體上分布情形 52 圖二、各 Xanthomonas 屬植物病原菌之 ITS 序列比對情形 53 圖三、以 IS1478 當探針，對以 EcoRI 或PstI 切割的染色體 DNA 進行南方雜配分析 55 圖四、以 IS1478 當探針，對選殖 7E23 及 7P23 的轉殖株質體以 dot blot 方式進行檢查分析 56 圖五、以IS1478 當探針進行南方雜配實驗，以確定圖四中具有 IS1478 雜配訊號之 F9 及 H1 所選殖的片段大小為正確的片段 57 圖六、以先前 IS1478 當探針進行南方雜配實驗，以確定圖四中具有 IS1478 雜配訊號之 7P23 片段之選殖株所選殖的片段大小為正確的片段 58 圖七、以 IS1478 當探針，挑選在 colony hybridization 中具有雜配訊號，選殖 1E43 片段的轉形株，抽取具有訊號之轉形株及周圍轉形株之質體，進行南方雜配分析 59 圖八、1E43 片段經定序後得到的結果 60 圖九、7P23 片段經定序後得到的結果 61 圖十、7E23 片段經定序後得到的結果 64 圖十一、Xcc ATCC33913 truncated NTPase 與 VirD4 蛋白之氨基酸序列比對結果 65 圖十二、使用 VirD4-F 及 VirD4-R 引子對以 PCR 方式增幅 Xcc ATCC33913、Xcc59、Xcc17、Xcc11，及 Xcc11A之 virD4 基因 66 圖十三、以 virD4 當探針，對分別以 EcoRI、HindIII，及 PstI 切割的 Xcc ATCC33913、Xcc59、Xcc17、Xcc11，及 Xcc11A 之染色體 DNA 進行南方雜配結果 67 圖十四、以 virD4 片段當探針，對分別以 EcoRI、SmaI，及 PstI 切割的 Xcc ATCC33913、Xcc17、Xcc11，及 Xcc11A 之染色體 DNA 進行南方雜配結果 68 圖十五、使用 VirB8-R 及 TypeIV-test 引子對以 PCR 方式增幅 Xcc ATCC33913、Xcc59、Xcc17、Xcc11，及 Xcc11A 的情形，以及選殖 Xcc17 及 Xcc11 之 PCR 片段後，定序比對之結果 70 圖十六、選殖以 virD4 當探針，在以 HindIII 切割的染色體 DNA 中，Xcc11 較 Xcc11A 多出的雜配訊號片段 71 圖十七、5H33-VgA1 片段之定性 72 圖十八、pSupSacAp- 質體之構築 75 圖十九、pSupSacAp-74::GMW 之構築 76 圖二十、pSupSacAp-VgA1::Kmr 之構築 77 圖二十一、分別以virD4、GmW，及 Kmr 當探針進行南方雜配試驗，以確定 Xcc17 及 Xcc11 突變株之正確性 78 圖二十二、測試各 Xcc17 第四型分泌系統表現抑制突變株及 Xcc11 vgA1 基因插壞株，胞外蛋白分泌的能力 79 圖二十三、對山東丸葉白菜小苗進行小苗戳莖實驗，以測試 Xcc17GMW-6 之致病能力 80 圖二十四、對山東丸葉白菜小苗進行小苗剪葉實驗，以測試 Xcc11Km-9 之致病能力 81 圖二十六、利用西方雜配分析，檢查第四型分泌系統表現抑制突變株 Xcc17GMW-6 及Xcc11-lee 能否將 OrfF’蛋白分泌至菌體外 8

Zinc Oxide Nanoparticle Disruption of Store-Operated Calcium Entry in a Muscarinic Receptor Signaling Pathway

The influences of ZnO nanoparticles on cellular responses to activation of muscarinic receptors were studied in Chinese hamster ovary cells expressing the human M3 muscarinic acetylcholine receptor. ZnO particles (20nm) induced cytotoxicity in a time and concentration-dependent manner: following a 24h exposure, toxicity was minimal at concentrations below 20 μg/ml but virtually complete at concentrations above 28 μg/ml. ZnO particles did not affect antagonist binding to M3 receptors or allosteric ligand effects, but increased agonist binding affinity while eliminating guanine nucleotide sensitivity. At a noncytotoxic concentration (10 μg/ml), ZnO increased resting [Ca2+]i from 40 to 130nM without compromising calcium homeostatic mechanisms. ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store-operated calcium entry (capacitive calcium entry). The latter effect was seen as (1) a decrease in the plateau phase of the response and (2) a decrease in Ca2+ entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50\u27s ≈ μg/ml). Thus, ZnO nanoparticles interfere with two specific aspects of the M3 signaling pathway, agonist binding and store-operated calcium entry

Morphology Control and Metallization of Porous Polymers Synthesized by Michael Addition Reactions of a Multi-Functional Acrylamide with a Diamine

Porous polymers have been synthesized by an aza-Michael addition reaction of a multi-functional acrylamide, N,N′,N″,N‴-tetraacryloyltriethylenetetramine (AM4), and hexamethylene diamine (HDA) in H2O without catalyst. Reaction conditions, such as monomer concentration and reaction temperature, affected the morphology of the resulting porous structures. Connected spheres, co-continuous monolithic structures and/or isolated holes were observed on the surface of the porous polymers. These structures were formed by polymerization-induced phase separation via spinodal decomposition or highly internal phase separation. The obtained porous polymers were soft and flexible and not breakable by compression. The porous polymers adsorbed various solvents. An AM4-HDA porous polymer could be plated by Ni using an electroless plating process via catalyzation by palladium (II) acetylacetonate following reduction of Ni ions in a plating solution. The intermediate Pd-catalyzed porous polymer promoted the Suzuki-Miyaura cross coupling reaction of 4-bromoanisole and phenylboronic acid