我々は有益な議論のためのMichelaさんデ·マルティーノ、ウィルフレッドR.ハーゲン、そしてKourosh Honarmand Ebrahimiに感謝しています。この作品はデルフト工科大学からの資金調達を起動によってサポートされていました。

1。 DPSの発現及び精製高純度のタンパク質を得ることがDNA保護アッセイのための必須の最初のステップである。 DPSタンパク質の精製は、4〜5日で行うことができる。 <ol><li> E.のプロテアーゼ欠損株を形質転換DPSタンパク質コード配列がクローニングされているにpETベクター（例えば、pET17など）を有する大腸菌 （例えばBL21（DE3）pLysSをなど）。 </li><li>ストリーク適切な抗生物質（例えば1.1で提供されていた例はアンピシリンとクロラムフェニコールなど）を含むルリア培地（LB）寒天プレートに細胞を変形させた。 37℃で一晩プレートをインキュベート℃に</li><li>適切な抗生物質を含むLB培地30ml中にプレートから単一コロニーを接種する。 200-250 rpmで振とうしながら37℃で一晩インキュベートする。 </li><li> 10ミリリットル一晩培養のそれぞれに適切な抗生物質を含む2×1 LのLB培地に接種。 OD 600に、振とうしながら、37℃で成長</約0.6のサブ&gt;。 （著しく最終生成物の純度を損なうことなく、より高いODの増加、タンパク質収量に細胞を成長は）0.3mMの濃度になるようにIPTGを添加しDPSの発現を誘導し、振とうしながら37℃で3-4時間、インキュベートする。 </li><li> 15分間6,000×gで遠心分離により細胞を収穫。誘導細胞培養物1L当たりDEAE緩衝液A（50mMのHEPES-KOH、pH7.5の、100mMのNaCl、0.1mMのEDTA）、7.5 mlの細胞ペレットを再懸濁する。サンプルを液体窒素中で凍結し、必要に応じて、-80℃で保存することができる。手順は、次に4℃で水浴中でサンプルを解凍することによって継続することができる</li><li>過剰発現DPSの劣化を防止するために細胞懸濁液に、プロテアーゼ阻害剤（例えば、カルビオケムセットIIIプロテアーゼ阻害剤、細胞懸濁液1mlあたり0.167μlの時）の混合物を加える。 </li><li>フレンチプレスを使用した無細胞抽出液を準備します。 20ミリリットルDEAEバッファ（連続フロー·モデルを使用している場合）、その後disrupと総理はフレンチプレスT 20 KPSI二回サンプル。不溶性粒子を明確にするために4℃で35分間30,000 xgでライセートを遠心分離し、上清を保存します。必要に応じて上清を、液体窒素を用いて凍結させ、-80℃で保存することができる。 </li><li> FPLCを用いて、DEAEバッファーで30ミリリットルDEAEセファロースCL-6Bカラムを平衡化。背景の上に0.2 MPaで最大圧力1〜2 ml /分でカラムを通してバッファを実行します。カラムに無細胞抽出液をロ ​​ードし、OD 280シグナルがベースラインを超えて増加し始めたら、フロースルーを収集し始める。 DEAEバッファーでカラムを洗浄し、連続フロースルーを収集しながら、OD 280値は、ベースラインに戻るまで。フロースルーは、結合したDNAから無料DPSタンパク質の大部分が含まれているはずです。 100％緩衝液B（50mMのHEPES-KOH、pH7.5で、1MのNaCl、0.1mMのEDTA）でカラムを洗浄します。この溶出液は、DPS-DNA複合体並びに他のタンパク質を含有し、廃棄することができる。 </li><li>広告を削除する硫酸アンモニウム沈殿を通じてditional混入タンパク質。プールされたフロースルーの音量を決定します。よく攪拌しながらゆっくり（10〜20分間かけて）4の62％飽和（溶液1mlあたり390 mg）を℃に乾燥小粒硫酸アンモニウムを追加します。完全な平衡を確保するために、硫酸アンモニウムの最後の部分を添加した後に、追加の20〜30分間混合物を攪拌する。混入タンパク質が溶液から析出する一方DPSは、可溶性のままになります。 </li><li> 4℃で30分間、20,000×gで遠心分離によって沈殿したタンパク質を除去°C、上清を保存する。 </li><li>徐々に94％飽和に達すると完全に平衡を確保するために20〜30分間撹拌し、上清の1ml当たり227ミリグラムの追加の硫酸アンモニウムを追加する。 DPSと、この高い塩濃度で沈殿し、4℃で30分間、20,000×gで遠心分離によって回収することができるDPSは、ペレットになり、上清を廃棄することができます。ペレットを-80℃で保存することができる°CであればデIRED。 </li><li>再懸濁バッファーでDPS含有ペレット（50mMのHEPES-KOH、pH7.5の、150mMのNaCl、0.1mMのEDTA）に再懸濁します。 2.5ミリリットルのサンプル量を調整します。 </li><li>緩衝液交換は、PD-10ゲル濾過カラムを使用して、硫酸アンモニウムを除去した。 25ミリリットルの再懸濁バッファーでゲルベッドを平衡化。 PD-10カラムの一番上にDPSサンプル2.5mlのを適用し、それがフロースルーを捨てインチ浸ることができます。 3.5ミリリットルの再懸濁緩衝液で溶出することでDPSを収集します。 </li><li> °C任意の不溶性成分を除去し、6〜10ミリリットルの希釈緩衝液（50mMのHEPES-KOH、pH7.5で、0.1mMのEDTA）で希釈することを保証するために4℃で10分間、16,000×gで緩衝液交換サンプルを遠心塩濃度はDPSがSPセファロースカラムに結合するのに十分に低い。 DPSの特に集中したサンプルは、液体の濁りによって証明が、完全に小さな天羽の添加により再溶解することができ、低塩バッファーに希釈すると溶解しにくくなることがあります5 M NaCl溶液（追加10〜20 mMの）のNT。 </li><li> SPバッファーA（50mMのHEPES-KOH、pH7.5で、50mMのNaCl、0.1mMのEDTA）と30ミリリットルSPロースファーストフローカラムを平衡化。背景の上に0.3メガパスカルの圧力限界で1.5〜2ミリリットル/分でカラムに通してバッファを実行します。カラムにサンプルをロードします。ベースラインにOD 280トレース戻るまでSPバッファーで洗浄し、フロースルーを捨てる。 2ミリリットルの画分を収集しながら、0〜100％の緩衝液Bから150ミリリットル直線勾配を実行します。 10％の変動が可能ですが、DPSは、約50％Bでの鋭いピークに溶出します。 </li><li> 15％SDS-PAGEゲル上の溶出画分を実行し、分光光度計を用いてOD 260 / OD 280の比率を測定することにより、DNAの混入を確認してください。ゲルはわずか19 kDaの周り一つのバンドを示すべきであり、OD 260 / OD 280が 0.7典型的には約です。最も純粋なDPS画分をプール。と遠心フィルターユニット（例えばアミコンウルトラ濾過ユニットなど）を使用10K分子量がDPSを集中し、ストレージ緩衝液（50mMのHEPES-KOH、pH7.5で、50mMのNaCl）にそれを交換するためにカットオフ。アリコート精製DPSし、-80℃で保管するための液体窒素で凍結℃の</li></ol> 2。 DNAプロテクションアッセイアッセイは、複数の時間に敏感な手順が含まれますし、再現性のある結果を得るために、第二にタイミングを合わせる必要があります。非常に多くの食材やピペット操作関与している、ピペッティングの表は、（ 表1を参照）のステップとボリュームを追跡することをお勧めします。アッセイに必要な総時間は3つ以上の時間ではありません。 <ol><li>密閉バイアルに30ミリリットルの水を測定し、溶液から酸素を除去するために10分（2注射針、流体内の1つの、その上のいずれかを使用）をN 2で水を洗い流す。 2 mMのストック溶液を得るために、水に0.0168グラムのFeSO 4•7H 2 Oを追加します。バイアルを密封し、混ぜた後、より多くの5分間窒素でフラッシュします。ソリューションでなければなりません明確な、黄色のいずれかのヒントが検出可能であれば、新たな鉄の溶液を調製する。 </li><li> 100mMのH 2 O 2溶液（9.2MH 2の10.87μlの水1ml中にO 2）を作る;氷上に維持し、アルミホイルで包むことによって遮光。自発的崩壊に起因する製造後約1〜2時間のためにのみ使用します。過酸化水素株式は、部分的に適切な保管（暗闇の中で、4℃）することにより改善することがあり故障、同様に対象となります。 OD 240を測定することにより、ストック濃度の定期的なチェックをお勧めします。 </li><li>室温の水浴中で急速にDPSの精製のアリコートを解凍し、氷上に保ち。沈殿凝集体テーブルトップpicocentrifugeで4,000×gで10秒間遠心します。分光光度計（OD 280 = 1.547 100μMモノマーDPSの濃度を示している）を使用して、遠心分離した後、濃度を測定する。 </li><li> 12倍反応バッファー（1を用いて高濃度在庫から線形DNAを希釈M MOPS-KOH pH7.0で、1 M NaCl）を100 ngの/μLの濃度に。直鎖状DNAを定量化を容易にするために使用されるが、プラスミドDNAを用いてもよい。 </li><li> PCRチューブにDNA緩衝液混合物のピペット1μlの。最終反応容積（マイナスSDS）を12μlのになるように反応に十分な水を追加します。この量は、ピペットテーブルを用いて予め算出されるべきである。 3μMの最終ドデカマー濃度にDPSを追加します。 DPSは、DNAに結合することを可能にするために室温で15分間インキュベートする。 </li><li>所望の濃度に達するために十分なの2mMのFeSO 4水溶液を加える。典型的な実験では、0から1 mMののFeSO 4の濃度の範囲を使用しています。高い濃度は、より広範なDNA分解の原因となりますが、また第二鉄の形成反応混合物中に沈殿につながる可能性があります。迅速100mMのH 2 O 2溶液（10mMの最終濃度）を1μlを追加し、フェントンを可能にするために5分間室温でインキュベートする場所を取るために媒介分解反応。 </li><li> 20％SDS（ドデシル硫酸ナトリウム）の0.8μLを加える。ミックスとDPS-DNA複合体を混乱させるための5分間85℃でインキュベートする。 SDSは、DPS-DNA複合体を不安定と定量のしやすさを向上させるDNAに対する不要なゲルシフトを防ぎます。オプションのステップは、非毒性生成物に触媒的分解過酸化水素によって反応を停止することである。このプロトコルで記述された条件については、このステップは、DNA分解の広い範囲を取得する必要がない。 </li><li>染料をロードする追加、1分間氷上でインキュベートし、アガロースゲルにロードします。ゲルを実行し、エチジウムブロマイドを用いたDNAのために染色。ゲルは、ゲル中のエチジウムブロマイドの配布に干渉するSDSを防止するために、ポスト電気泳動染色したのではなく、電気泳動の前になければなりません。 </li></ol>

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我々は、開示することは何もありません。

この記事で説明したようにDPSの精製プロセスは非常に堅牢です。純度は一貫して（&gt; 99％）高くなっている。他のタンパク質は、可視バンドとしてSDS-PAGEゲル上で表示されません。 DPSの非常に高い濃度でインキュベートしたときに、部分的DNA分解によって証明されるようにもかかわらず、いくつかの精製されたDPSバッチは、ヌクレアーゼ活性を有するように見える。これは、我々が浄化を通...

好気性生物は常にそれらのDNAだけでなく、他の重要な生体高分子を損傷する可能性が活性酸素種と競合しなければなりません。酸化的損傷の毒性を中和する一つの強力なツールは、飢餓細胞（DPS）からDNA結合性タンパク質である。飢えE.から1992年の発見以来大腸菌培養1、DPSは、細菌および古細菌2以上の300種で確認された。固定相の間のDPSの大量アップレギュレーションは、Eの最も高度に発現核様体関連タンパク質になります飢餓条件3、4アンダー大腸菌 。また、DPSは飢餓、高鉄濃度、UV光照射、熱ショック、および酸化ストレス5,6を含む多くの様々なストレスの間、細菌の生存率およびDNAの完全性の両方を維持することが示されている。 12モノマー、Wの安定したホモオリゴマー複合体へ構造的には、DPSは、自己関連付けHICHは、球状の中空シェルに組み立てる。 〜4.5 nmの全体の内部空洞は、小分子7の通過を可能に細孔を介して溶剤外部からアクセス可能であり、例えば鉄8石灰化などの金属を封鎖することができる。 DPSの保護効果は、非特異的DNA結合1、フェロキシダーゼアクティビティ、および鉄の貯蔵8を含むそのいくつかの生化学的活動から派生。 DPSの有益な生化学的な活動の詳細な研究では、最初にその精製を必要とします。 DPSは、だけでなく、他のタンパク質から分離するだけでなく、任意の結合したDNA 7からしなければならないとしてDPSの精製は、精巧な手順です。我々の最適化された精製プロセスは、2つのイオン交換カラムおよび硫酸アンモニウム沈殿工程からなる、多くの一般的な技術を使用する。高濃度のDPSが低塩条件下で溶液から析出ことができるように複数のバッファ交換が必要である。一度DPSタンパク質は、精製された、それは直接フェロキシダーゼ活性8、DNA結合化学量論9および10の鉄結合機構を測定するアッセイに適用することができる。精製されたDPSは、他の潜在的なアプリケーションを持っています。 DPSの安定した中空球状の構造は、12小説磁性ナノ粒子を合成するタンパク質キャビティ11内でも、反応室として疎水性粒子を格納するための足場として使用されている。 活性酸素種による損傷を媒介するDPSの防御能は明らかになり、直接DNA保護アッセイ13,14を用いて実証することができる。鉄はフェントン化学介してH 2 O 2分解を触媒するときに、このインビトロ手順において 、ラジカル種が生成される。これらのラジカルは直接反応に存在するDNAに損傷を与え、完全に高濃度で、それを分解することができます。 2つの重要なDPS活動は両方直接クエン酸フェンタニルの影響を打ち消すことが上の媒介ラジカル生産。 DPSは、プロセスで使用可能過酸化水素を消費し、石灰化触媒を介して鉄濃度を低下させる。さらに、DNAへの結合DPSは、潜在的にラジカルによるダメージから物理的にシールドと反応性の低い表面積の小さい容積にそれを凝縮することができる。これら二つの特性の組み合わせがよく、保護DPS活性を測定する目的に適したDNA保護アッセイを行う。 DNA保護アッセイは、非常に汎用性があり、DPS特性を越えて様々な用途に用いることができる。ラジカルによる損傷は、細胞内の応力の一般的な形態であり、多くの異なるタンパク質および化学物質は、それを打ち消すために使用される。ラジカルによる損傷のマーカーとしてDNAの完全性を用いたアッセイの一般的原理は、ほぼすべてのラジカル生成反応または反作用剤と組み合わせて使用​​することができる。中でも、アッセイは正常に抗酸化特性を決定するために使用されているK.の食品産業15で使用するためのカスミソウエキス、水酸基被害調停16日に尿酸の効果を特徴付けるため、そして毛皮転写調節蛋白質17の機能に新たな洞察を得るために。 論文におけるアッセイの多くの用途にもかかわらず、我々は初めて、多くの研究者のために不必要に骨の折れるプロセスについてのアッセイを設定するなりした、多くの最適化とトラブルシューティングの手順が必要であったことがわかった。我々はこの記事では、この議定書は、エントリのこの障壁を取り除くことを目指しています。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>BL21(DE3)pLysS competent cells</td>
 <td>Promega</td>
 <td>L1195</td>
 </tr>
 <tr>
 <td>pET-17b DNA</td>
 <td>EMD-Millipore</td>
 <td>69663-3</td>
 </tr>
 <tr>
 <td>LB broth powder</td>
 <td>Sigma-Aldrich</td>
 <td>L3022</td>
 </tr>
 <tr>
 <td>Ampicillin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>A0166</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C0378</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td>I6758</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>BDH</td>
 <td>441476L</td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>Merck</td>
 <td>105033</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>VWR</td>
 <td>443824T</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 </tr>
 <tr>
 <td>Protease Inhibitor Cocktail Set III</td>
 <td>EMD-Millipore</td>
 <td>539134</td>
 </tr>
 <tr>
 <td>DEAE-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>DFF100</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>A4418</td>
 </tr>
 <tr>
 <td>PD-10 Desalting Columns</td>
 <td>GE Healthcare LS</td>
 <td>17-0851-01</td>
 </tr>
 <tr>
 <td>SP-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>S1799</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centr. Filter (10K MWCO)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>Ferrous Sulphate heptahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>F8048</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide solution</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Calbiochem</td>
 <td>475898</td>
 </tr>
 <tr>
 <td>SDS solution</td>
 <td>Bio-Rad</td>
 <td>161-0418</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Sigma-Aldrich</td>
 <td>E1510</td>
 </tr>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Static incubator</td>
 <td>Hettich</td>
 <td>INE500</td>
 </tr>
 <tr>
 <td>Shaking Incubator </td>
 <td>New Brunswick Sc.</td>
 <td>Inova 44</td>
 </tr>
 <tr>
 <td>Cooled centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-E</td>
 </tr>
 <tr>
 <td>Table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5424</td>
 </tr>
 <tr>
 <td>Cell disrupter</td>
 <td>Constant Systems Ltd.</td>
 <td>TS2/40/AA/AA</td>
 </tr>
 <tr>
 <td>FPLC Purifier</td>
 <td>General Electric</td>
 <td>AKTA</td>
 </tr>
 <tr>
 <td>Airtight vials</td>
 <td>Cole-Parmer</td>
 <td>EW-08918-85</td>
 </tr>
 <tr>
 <td>Syringe needles</td>
 <td>BD</td>
 <td>305128</td>
 </tr>
 <tr>
 <td>Pipettes</td>
 <td>Eppendorf</td>
 <td>Z683779-1EA, Z683795-1EA</td>
 </tr>
 </tbody>
</table>

application of an in vitro dna protection assay to visualize stress mediation properties of the dps protein

酸化ストレスは好気性の生命の避けられない副産物である。酸素分子は、地上代​​謝に不可欠ですが、それはまた、生物内の多くの有害な反応に参加しています。生活のためのもう一つの重要な化合物であり、好気的代謝と鉄の組み合わせは、フェントン化学を通じてラジカルを生成し、細胞成分を分解するのに十分です。 DNA修復がはるか些細からそのままDNA分解は、間違いなく細胞内のラジカルを含むほとんどの有害なプロセスである。この記事で紹介したアッセイは、ラジカル媒介DNA損傷の分子と酵素の効果を測定し、可視化する定量的な手法を提供しています。 DNA保護アッセイは、タンパク質や化学物質の保護特性のin vitroでの特性のために、シンプルで迅速、かつ強力なツールです。それは有害な酸化反応にDNAをさらすと関心の化合物の種々の濃度を加算関与。化合物濃度の関数としてのDNA損傷の減少または増加は次いで、ゲル電気泳動を用いて可視化される。本稿では飢餓細胞（DPS）からDNA結合蛋白質の保護特性を測定することにより、DNA保護アッセイの手法を示す。 DPSは力強く環境ストレスに対抗するために300以上の細菌種によって利用されるミニフェリチンです。ここでは、DPS精製プロトコルとDPSによってDNA保護を評価するための最適化されたアッセイ条件を提示する。

ここで説明DPSの精製プロセスは非常に再現可能である。 E.のL 2を用いて説明プロトコールに従って精製DPSと、出発点として、 大腸菌培養 、一般的に5〜12μMの濃度で12 DPSを含むタンパク質の2.5ミリリットルが得られます。長い誘導時間（4時間）は、このばらつきを軽減するように見える。 SDS-PAGEゲル（ 図1）から明らかなようにタンパク質の純度は99％以上?...

Watch this Scientific Journal Video about Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein at JoVE.com

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

飢餓細胞（DPS）からDNA結合タンパク質は、細菌のストレスとの闘いにおいて重要な役割を果たしている。この記事での精製について説明<em&gt; E.大腸菌の</em&gt; DPSとするためのプロトコル<em&gt; in vitroで</em&gt;アッセイは、活性酸素種による分解からのDNAのDPS媒介性保護を示す。

の適用<em&gt;インビトロ</emDPSタンパク質のストレス調停プロパティを可視化するために&gt; DNA保護アッセイ

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

application-an-vitro-dna-protection-assay-to-visualize-stress

Research

JoVE Journal

Biology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

11.1K ビュー.  Delft University of Technology. 飢餓細胞（DPS）からDNA結合タンパク質は、細菌のストレスとの闘いにおいて重要な役割を果たしている。この記事での精製について説明 E.大腸菌の DPSとするためのプロトコル in vitroでアッセイは、活性酸素種による分解からのDNAのDPS媒介性保護を示す。

ビデオ: Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

アン試験管内でフルオロブロックの腫瘍の浸潤アッセイ

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

生物学

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

DNA修復タンパク質の作用のメカニズムを調べるためにストップトフロー速度論の手法の適用

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

相間細胞核の力学特性を探査する生物物理学的なアッセイ：基板の歪みのアプリケーションおよびマイクロニードル操作

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

タンパク質アルギニンメチル化を研究するためのin vitroでメチル化アッセイで

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

全身酸化ストレスの間接的な指標として、コメットアッセイ

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

慢性後のマウス脾細胞におけるDNA損傷と修復の測定<em&gt;インビボ</em&gt;β-およびγ-放射線の非常に低い用量への暴露

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

「せん断リング」の組み立てと応用：小説内皮軌道、単方向および定期的な流動のためのモデルとせん断応力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

パインウッドセンチュウの交配挙動の観察と定量化、<em&gt;マツノザイセンチュウ</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...