この研究は、基礎科学研究プログラムによって支えられた (プロジェクト番号: 2016R1A2B4013467) を通じて国立研究財団の韓国 (NRF) 科学省 ICT ・将来計画 2016 によって資金を供給し、韓国基礎科学でもサポートされています。研究プログラム研究所 (プロジェクト番号: C36707)。

1. DMMTAVの合成
<ol><li>
化学準備とモル比混合 DMAVナ2S、H2SO4 注: DMAV: Na2S:H24 = 1:1.6:1.6<ol>
<li>40 mL の脱イオンと N2の DMAVの 5.24 g を溶解-パージ (少なくとも 30 分はパージ) 50 mL 遠心管中の水。</li>
		<li>Na2S·9H2O で 50 mL の脱イオンと N2の 14.41 g を溶解することにより Na2S 試薬を準備-250 mL フラスコに水を削除します。</li>
		<li>準備 H2純水 40 mL に濃硫酸 (96%) の 3.3 mL を追加して4試薬だと N2-50 mL の遠心管中の水をパージします。 注: DMAVの最終的なモル比: Na2S:H2SO4 = 1:1.6:1.6 1,3,4,8,15。</li>
		<li>DMAVソリューション (ステップ 1.1.1) の準備 40 mL を 250 mL のフラスコ (ステップ 1.1.2) に含まれる Na2S 溶液 50 mL に追加します。DMAV 10 mL のパージされた水で管内を洗浄し、250 mL フラスコを同様にこれを追加します。</li>
		<li>ガラス管が装備 3 つ穴ゴム栓付けフラスコを閉じます。ガラスを使用して、N2ガス流入、流出と H2SO4液入口それぞれチューブします。フラスコを閉じた直後にフラスコに N2ガスの流れを許可します。 注: 反応液の表面に飛散することがなく流れにガス圧を維持しなければなりません。</li>
		<li>耐酸性チューブ H2のガラス チューブにその4液入口 H2の準備の 40 mL を追加しに 50 mL 注射器を接続4ソリューション (1.1.3 ステップ)。H2の 40 mL を追加し、4ソリューションでは、ゆっくりと段階的に。 注意:硫酸を追加するには、時に白い煙が生成されます。換気ヒューム フードを使用します。</li>
		<li>定期的に硫酸を加えている間フラスコ内で反応混合物の色の変化を監視します。だから、H2の 4-5 mL の間隔が値下がりしましたを維持4;混合物は、白い曇りのソリューションにする必要があります。 注: 急速な濃硫酸添加による瞬時の黄色い沈殿物があります。</li>
		<li>1.1.4 のステップの始まりから 1 時間立っている反応液を確認します。</li>
	</ol>
</li>
	<li>
DMMTAV抽出液-液抽出法を用いて
	<ol>
<li>1 時間後に、ジエチル エーテルの約 200 mL を含む, 漏斗に反応液を注ぐ。</li>
		<li>5-10 分、活栓を切り替えることによって数回ガスを放出の漏斗を振る。 注: 合成 DMMTAVはジエチル エーテル (0.713 g·mL-1) の上位層に転送されます。 注意:ジエチル エーテルのガスは有害かもしれない換気ヒューム フードを使用します。</li>
		<li>、ビーカーに反応液を収集し、個別にボトルの DMMTAVを含むジエチル エーテルを収集します。同じ, 漏斗に反応液を置き、reshaking の新鮮なジエチル エーテルの約 200 mL を追加します。1.2.2 - 1.2.3 の手順を 3 回繰り返します。</li>
		<li>もう一度、同じ, 漏斗にステップ 1.2.3 から収集したジエチル エーテルを注ぐし、約 100 mL の N2を追加-脱イオン水を削除します。5-10 分のために振るし、N2破棄-純水と純度のためジエチル エーテルの数 mL をパージします。ガラス シャーレ (160 mm の最小内径) と最小の高さ 50 mm で残りのジエチル エーテルを収集します。</li>
		<li>種変換を防ぐために N2雰囲気グローブ ボックスにガラス シャーレを転送します。 注意:パス ボックスのコンセントを通じて真空ポンプに溶媒を抜けないことを確認します。</li>
		<li>Dimethylmonothioarsinate の白色沈殿物まで乾燥 (結晶化 DMMTAV) は、ガラス シャーレに形成されます。 注: プロトコルはここで一時停止することができます。</li>
	</ol>
</li>
	<li>
合成 DMMTAVおよび記憶域の検証
	<ol>
<li>DMMTAVの結晶の白色沈殿物を取るとを測定し、総重量を記録します。 注意:硫化水素ガスを吸入しないように換気の良いヒューム フードやグローブ ボックスを使用します。</li>
		<li>ディゾルブ N2の 50 mL の DMMTAVを結晶化-脱イオン水をパージ、0.2 μ m のシリンジ フィルターを介して黄色の沈殿物をフィルター処理します。</li>
		<li>現在使用されている DMAV合計は DMMTAV、すなわち≈9、649 mg As· に変換されますと仮定L-1。DMMTAV ≈1 mg· に原液を希釈します。L-1と ≈40 µg·エレクトロ スプレー イオン化を用いた検証解析 L-1質量 Spectromtery (MS)、高速液体クロマトグラフィー-ICP-MS、それぞれ。</li>
		<li>DMMTAV m/z 155 ポジティブ イオン モードで、 m/zマイナス イオン モード (表 1) で 153 MS11,16とフラグメントを使用してのm/zを分析します。 注: m/z (表 1) の参照値を参照してください。</li>
		<li>適切な溶離液条件による高速液体クロマトグラフィー-誘導結合プラズマ質量11,16,17原液の DMMTAVのクロマト グラムを分析および確認に記載されている保存時に大きなピークが検出、文学。 注: 合成 DMMTAVの純度は、1.3.5 のステップから解析結果を用いた計算する必要があります。</li>
		<li>酸分解11後 ICP-MS を用いた総砒素濃度を分析し、合成 DMMTAV原液希釈倍率と次式のように純度を使用して真の DMMTAVの濃度を計算します。 濃度 (µg· として分析された合計L-1) ·希釈係数 ·純度 (%) = True DMMTAV DMMTAV原液 (µg· 濃度L-1)</li>
		<li>暗闇のなかでさらに定量的なスペシエーション分析18DMMTAV 4 ° C で原液を保存します。</li>
	</ol>
</li>
</ol>DMDTAVの合成
<ol><li>
化学準備とモル比混合 DMAVナ2S、H2SO4 注: DMAV: Na2S:H24 = 1:7.5:7.5<ol>
<li>40 mL の脱イオン水 50 mL 遠心管中の DMAVの 1.38 g を溶解します。</li>
		<li>ナ2250 mL フラスコ 50 mL の脱イオン水 S·9H2O の 18.01 g を溶解することにより Na2S 試薬を準備します。</li>
		<li>40 mL の脱イオン水に濃硫酸 (96%) の 4 つの mL を追加して4試薬は 50 mL の遠心管に含まれるように H2を準備します。 注: DMAVの最終的なモル比: Na2S:H2SO4 = 1:7.5:7.51,3,4,8,15。</li>
		<li>DMAVソリューション (ステップ 2.1.1) の準備の 40 mL を 250 mL のフラスコ (ステップ 2.1.2) に含まれる Na2S 溶液 50 mL に追加します。DMAV 10 mL の脱イオン水で管内を洗浄し、250 mL フラスコを同様にこれを追加します。</li>
		<li>H2の準備の 40 mL を追加し、4ソリューション (2.1.3 ステップ)、ゆっくりとフラスコに、段階的に。</li>
		<li>定期的に硫酸を加えている間フラスコ内で反応液の色の変化を監視します。だから、H2の 4-5 mL の間隔が値下がりしましたを維持4;混合物は、白/黄色曇りソリューションをする必要があります。 注: 急速な濃硫酸添加により瞬時の黄色い沈殿物があります。 注意:硫酸を追加するには、時に白い煙が生成されます。風通しヒューム フードを使用します。</li>
		<li>カバーなし一晩フラスコ反応液を維持します。 注: プロトコルはここで一時停止することができます。</li>
	</ol>
</li>
	<li>
DMDTAV抽出固相抽出 (SPE) を用いた
	<ol>
<li>一晩立って反応後 C18 シリンジを使用して反応液をトラップするためにシリカ系の SPE の入力フィルターは合成樹脂に DMDTAVです。 注意:換気ヒューム フードを使用します。</li>
		<li>10 mM 酢酸アンモニウム (pH 6.3) を準備するには、1 L の脱イオン水に酢酸アンモニウム 0.77 g を溶解します。10 mM 酢酸アンモニウムの十分な量を溶出吸着 DMDTAVを抽出する C18 シリンジ (ステップ 2.2.1) を介して。ガラス シャーレ (160 mm の最小内径) と最小の高さ 50 mm でフィルター処理されたアンモニウム酢酸を収集します。</li>
		<li>種変換を防ぐために N2雰囲気グローブ ボックスにガラス シャーレを転送します。 注意:パス ボックスのコンセントを通じて真空ポンプに溶剤を抜けないことを確認します。</li>
		<li>Dimethyldithioarsinate の白色沈殿物まで乾燥ガラス シャーレに (結晶化 DMDTAV) を結成します。 注: プロトコルはここで一時停止することができます。</li>
	</ol>
</li>
	<li>
合成 DMDTAVおよび記憶域の検証
	<ol>
<li>結晶 DMDTAV、白色の沈殿物を取るし、総重量は、測定の記録を測定します。 注意:硫化水素ガスを吸入しないように換気のヒューム フードやグローブ ボックスを使用します。</li>
		<li>ディゾルブ N2の 50 mL の DMDTAVを結晶化-パージされた脱イオン水 50 mL にチューブを遠心し、0.2 μ m のシリンジ フィルターを介して任意の沈殿物をフィルター処理します。</li>
		<li>現在使用されている DMAV合計は DMDTAV、すなわち≈2 539 mg As· に変換されると仮定します。L-1。DMDTAV ≈1 mg· に原液を希釈します。L-1と ≈40 µg·それぞれ MS と高速液体クロマトグラフィー-ICP-MS を用いた検証解析 L-1 。</li>
		<li>DMDTAV m/z 171 ポジティブ イオン モードで、 m/zマイナス イオン モード (表 1) で 169 ESI MS11,16とフラグメントを使用してのm/zを分析します。 注: m/z (表 1) の参照値を参照してください。</li>
		<li>適切な溶離液条件による高速液体クロマトグラフィー-誘導結合プラズマ質量11,16,17原液の DMDTAVのクロマト グラムを分析し、「保持時間ピークがあることを確認文学。 注: 合成 DMDTAVの純度は、2.3.5 のステップから解析結果を用いた計算する必要があります。</li>
		<li>酸分解11後 ICP-MS を用いた総砒素濃度を分析し、次の式として希釈倍率と純度を使用して合成の DMDTAV在庫ソリューションで真の DMDTAV濃度を計算します。 濃度 (µg· として分析された合計L-1) ·希釈係数 ·純度 (%) = True DMDTAV DMDTAV原液 (µg· 濃度L-1)</li>
		<li>暗闇のなかでさらに定量的なスペシエーション分析18DMDTAV 4 ° C で原液を保存します。</li>
	</ol>
</li>
</ol>

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<li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arsenic+speciation+in+the+blood+of+arsenite-treated+F344+rats%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">Arsenic speciation in the blood of arsenite-treated F344 rats.</a> Chem. Res. Toxicol. 26, 952-962 (2013).</li>
<li>Alava, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HPLC-ICP-MS+method+development+to+monitor+arsenic+speciation+changes+by+human+gut+microbiota%5BTitle%5D)+AND+%22Biomed.+Chromatogr%22%5BJournal%5D)">HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota.</a> Biomed. Chromatogr. 26, 524-533 (2012).</li>
<li>Kurosawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+metabolic+activation+associated+with+glutathione+in+dimethylmonoarsinic+acid+%28DMMTAV%29-induced+toxicity+obtained+from+in+vitro+reaction+of+DMMTAV+with+glutathione%5BTitle%5D)+AND+%22J.+trace+Elem.+Med.+Biol%22%5BJournal%5D)">A novel metabolic activation associated with glutathione in dimethylmonoarsinic acid (DMMTAV)-induced toxicity obtained from in vitro reaction of DMMTAV with glutathione.</a> J. trace Elem. Med. Biol. 33, 87-94 (2016).</li>
</ol>

著者が明らかに何もありません。

開発されたプロトコルが重要なステップ、前研究1,3,4,8,15省略または省略する難しさまたは中の障害になった可能性を明らかにDMMTAV DMDTAVの合成。DMMTAVは酸化に敏感な1、5その合成のための化学試薬調製 N...

ジメチルアルシン酸 (DMAV)、急性毒性とメチル化と摂取1,2時なチオール化による遺伝毒性の両方を展示する実証されているなチオール化ヒ素の代謝経路は集中的にされて学びの in vitroとin vivoの両方3,4同様環境メディア (例えば、埋め立て地の浸出水)5,6のように。以前の研究で発見した両方が減少し、リビングの DMAVのチオール類縁体細胞、dimethyldithioarsinic 酸 (dimethylmonothioarsinic 酸 (DMMTAV)、例, dimethylarsinous 酸 (DMAIII)DMDTAV)7,8,9, 欠損 thioarsenicals DMMTAV他の無機または有機ヒ素10知られているより大きい毒性の展示など。猛毒の thioarsenicals の豊富さは深刻な環境影響を以来、彼らは人間および高い硫化水素の条件11の下で環境に危険をもたらす可能性があります。ただし、DMMTAV DMDTAV (トランス) 形成と環境中運命のメカニズムもさらなる研究が必要です。したがって、thioarsenicals の定量分析は、DMMTAV DMDTAVの環境への影響の理解を改善するために必要です。
標準的な化学物質は、定量分析の重要な条件です、DMMTAV DMDTAVの基準が他の種に種変換の高危険のための先行研究を複製することによって得ることが困難と要員の合成手順12。さらに、参照されるメソッドには、標準化学物質を合成し、定量分析を実行する実用的な困難につながる可能性があります制限があります。DMMTAV DMDTAV一般的で一定モル比1またはバブリング H2S4ガス DMAV 13, 14のソリューションを通じて、DMAVナ2S、H2を混合して用意しています。酸素の毒性が高く経験の浅いユーザーの制御が困難な H2S ガスの直接供給を使用して硫黄のバブリング法機能置換。逆に、上記混合法1DMMTAV DMDTAV環境研究5,6,12の定性分析に広く使用機能のなチオール化H2S のため直接と比較して4 DMMTAV DMDTAV、対象化学物質を生成する化学量論的制御を簡単に作り出す Na2S と H2を混合することによって生成された DMAVH2S ガスを使用します。
つながる可能性があります彼らの重要な実験手順のいくつかでこの研究展示の制限に記載されている混合法手順1,3,4,8,15参照実験に失敗しました。たとえば、特定溶媒 (すなわち、脱イオン水) 準備と抽出の詳細と合成のヒ素の結晶化が過剰省略または十分な細部で記述されていません。このような分散し、手順についての限られた情報が thioarsenicals と信頼性の定量化分析の一貫性のない形成につながる可能性があります。したがって、ここに開発された変更されたプロトコルは、DMMTAVと DMDTAV定量的種の分離分析と原液の合成について説明します。

<table><tbody><tr><td>Cacodylic acid</td><td>Sigma-Aldrich</td><td>20835-10G-F</td><td></td></tr><tr><td>Sodium sulfide nonahydrate</td><td>Sigma-Aldrich</td><td>S2006-500G</td><td></td></tr><tr><td>Sulfuric acid 96%</td><td>J.T.Baker</td><td>0000011478</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A7262-500G</td><td></td></tr><tr><td>Formic acid 98%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>066-00461</td><td></td></tr><tr><td>Diethyl ether (Extra Pure)</td><td>Junsei Chemical</td><td>33475-0380</td><td></td></tr><tr><td>Adapter cap for 60 mL Bond Elut catridges</td><td>Agilent Technologies</td><td>12131004</td><td>Syringe type of SPE</td></tr><tr><td>Bond Elut C18 cartridge</td><td>Agilent Technologies</td><td>14256031</td><td>Syringe type of SPE</td></tr><tr><td>HyPURITY C-18</td><td>Thermo Scientific</td><td>22105-254630</td><td>5 um, 125 x 4.6 mm</td></tr><tr><td>Glovebox</td><td>Chungae-chun, Rep. of Korea</td><td></td><td>Customized&amp;nbsp;</td></tr><tr><td>Agilent 1260 Infinity Bio-inert LC</td><td>Agilent Technologies</td><td>DEAB600252, DEACH00245</td><td></td></tr><tr><td>Agilent Technologies 7700 Series ICP-MS</td><td>Agilent Technologies</td><td>JP12031510</td><td></td></tr><tr><td>Finnigan LCQ Deca XP MAX Mass Spectrometer System</td><td>Thermo Electron Corporation</td><td>LDM10627</td><td></td></tr></tbody></table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

ジメチルアルシン酸 (DMAV) なチオール化の代謝経路によって生成される、dimethylmonothioarsinic 酸 (DMMTAV) などの欠損 thioarsenicals および dimethyldithioarsinic 酸 (DMDTAV) は最近ずっと環境だけでなく、人間の臓器で発見します。欠損 thioarsenicals と環境メディアの安定性の生態学的影響を判断する DMMTAV DMDTAVを定量化することができます。これらの化合物の合成法は標準化され、挑戦的な先行研究を複製することです。さらに、化合物種変換なしのストレージを含むストレージの技術についての情報の欠如があります。また、合成法について限られた情報のみが使用できる標準化学物質を合成し、定量分析を実行するのに実験的な困難がある可能性があります。ここに提示されたプロトコル欠損 thioarsenicals、DMMTAV DMDTAV、実質的に変更された合成方法を提供し、高速液体を使用して種の分離分析の定量化に役立つ誘導結合プラズマ質量分析法 (高速液体クロマトグラフィー-ICP-MS) と組み合わせてクロマトグラフィー。化学試薬、ろ過方法、ストレージの準備に焦点を当てたこの手順の実験の手順が変更されました。

DMMTAVは、誤って DMAIII合成法19によって準備されている、合成 DMMTAV DMDTAVの検証は合成、抽出と理想的な基準を決定するための重要なステップ化学物質。合成化学物質はピークの DMMTAV (MW 154 g·mol-1)、エレクトロ スプレー イオン化質量のいずれかの正または負のイオン モードを使用して DMDTAV (17...

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

DMMTAV DMDTAV環境用 DMAVを使用しての作製: 合成、精製、および確認

本稿では dimethylmonothioarsinic 酸 (DMMTAV) の実験的プロトコルの修正と dimethyldithioarsinic 酸 (DMDTAV) の合成、DMAV の混合によってジメチルアルシン酸 (DMAV) なチオール化を誘導します。、Na2S と H24。修正されたプロトコルは、それにより定量分析における実験的障害を引き起こしている可能性が合成手順の制限の克服、実験ガイドラインを提供します。

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

preparation-dmmtav-dmdtav-using-dmav-for-environmental-applications

Research

JoVE Journal

Environment

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

5.5K ビュー.  Korea Basic Science Institute, Seoul Center. 本稿では dimethylmonothioarsinic 酸 (DMMTAV) の実験的プロトコルの修正と dimethyldithioarsinic 酸 (DMDTAV) の合成、DMAV の混合によってジメチルアルシン酸 (DMAV) なチオール化を誘導します。、Na2S と H24。修正されたプロトコルは、それにより定量分析における実験的障害を引き起こしている可能性が合成手順の...

ビデオ: Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

水中での超分子ポリマーのサイズ、形状と安定性を制御する

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

工学

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

化学

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

1分子蛍光イメージングのためのコンパクトな量子ドット

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

新生児低酸素症の生化学的測定

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

医学

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

調製と<em&gt;インビトロ</em&gt;磁気共鳴イメージングのためのデンドリマーベースの造影剤のキャラクタリゼーション

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

P- ニトロ安息香酸の存在の連続流れ化学: 反作用

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

ブラシのような反応性表面を加工し、アズラクトン官能基化ブロック共重合体の架橋膜

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

核磁気共鳴分光法によるアルギニンメチロームの探索

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

がん研究

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

チオバルビツール酸反応性物質アッセイを用いた生体試料における酸化ストレスの評価

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

ヘッドスペースGC-MSによる動物由来医薬品のトリメチルアミン検出

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

著者スポットライト:ヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析による動物由来医療におけるトリメチルアミン検出の改良技術  

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

ヘッドスペースガスクロマトグラフィー-タンデム四重極質量分析法による動物由来医薬品におけるトリメチルアミン検出の改良技術

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

DMMTA^V DMDTA^V環境用 DMA^Vを使用しての作製: 合成、精製、および確認

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