PAは、ラバル大学の医学部からの奨学金の受取人 &quot;ピエール·デュラン」です。ポリソーム精留塔をRMに自然科学とカナダの工学研究会（MOP-CG095386）によってサポートされていたこの作品は、RMR Mにイノベーション助成金のためのカナダの財団（MOP-GF091050）を通じて取得した新しいCIHR研究者の給与賞を保持している。 私たちは、博士に感謝しています。有益な助言のためのE. Khandjian、I. Gallouzi、S.ジ - マルコとA. Cammas。

プロトコルは、ガイドラインに従っています ラバルの倫理審査委員会により承認されました。 1。細胞培養の準備と脳の操作<ol><li>哺乳類とショウジョウバエ細胞<ol><li>アメリカン·タイプ·カルチャー·コレクションが推奨するようにしたHeLa子宮頸癌細胞とシュナイダーショウジョウバエの胚細胞を増殖させる。低継代の細胞を操作します。 </li><li> 80％の集密度の実験日に到達するために、プレートの細胞。最良の結果を得るには、各実験条件についての細胞の〜12×10 6を使用しています。ポリソーム分析のための抽出物を加える前に、少なくとも90分間、新鮮な完全培地を添加することにより翻訳を刺激する。 </li></ol></li><li>脳の分離：生後9〜16日の間、老齢マウスの脳を分離します。安楽死は、麻酔および頸椎脱臼した後、CO 2吸入を必要とする。犠牲に続いて、脳全体のどちらかの場所を隔離I-80℃でTまたは直接すぐに使用冷PBS 1Xにそれを転送します。 </li></ol> 2。密度勾配分画系の製造に<ol><li> 5分間、0.1％SDSで配管システムを洗浄します。 </li><li> DEPC水で洗浄する前に、5分間RNaseZAPソリューションで、その後、5分間、70％エタノールでチューブシステムを洗浄します。 </li><li>装置の配管系を乾燥させるために空気をポンプ。 </li></ol>ショ糖勾配の3。準備<ol><li> 20mMのトリス-HCl、pH7.4、1.25のMgCl 2、150mMのNaCl、および1mM DTT、15％、55％ショ糖溶液を作る。 </li><li>生成する 製造業者の使用説明書に記載されているように、前者イスコモデル160勾配を用いて15％から55％線形スクロース勾配。しかし、勾配の気泡や乱流の生成を避けるために、周知TRIS蠕動ポンプの速度を制御することが重要である。使用するまで4℃で勾配を維持する。 </li><li>勾配メーカープログラムが実行されている時間の間、4℃で超遠心機を冷却する</li></ol> 4。細胞の作製とマウスの脳の抽出物<ol><li>細胞抽出物の準備<ol><li>氷と洗浄細胞の上に置いてプレート（S）は、冷PBS 1Xで3回。 </li><li>補充した溶解緩衝液1ml（pH7.4の20mMトリス塩酸、1.25のMgCl 2、150mMのNaCl、1mMのDTT、1％ノニデットP40、5 U / mlのRNase阻害剤、収穫細胞（〜12×10 6個）完全なミニEDTAフリープロテアーゼ阻害剤カクテル錠付き）、エッペンドルフチュー​​ブに転送し、1ccののU100インスリン注射器28 G 1/2を介してそれらを15Xを渡すことでよく混ぜる。細胞溶解物を氷上で15分間休憩しましょう​​。 </li><li> 10×対物レンズを用いた位相差顕微鏡で観察することにより細胞溶解を評価するためにスライド上に細胞溶解物を数滴入れた。唯一の核は見えるはずず、細胞膜は、細胞溶解のために証明する見えないはずです。対照として、観察未溶解細胞。 </li><li> 4℃で20分間、11,000×gでの遠心分離によって細胞溶解物を明確にし、ポリリボソームを含む可溶性ライセートを保つ。 </li></ol></li><li>マウス脳抽出物の調製<ol><li>溶解緩衝液2mlの氷冷Dounceホモジナイザーで10ストロークにより単離された脳全体（〜500mg）をホモジナイズし、4℃で15分間、11,000×gで遠心分離することによってホモジネートを明らかにする</li><li> 50％ショ糖のクッションの上に得られた上清をロードし、4℃で、TH-641超遠心ローターを用い20万×gで2時間沈降を進める</li><li>溶解緩衝液1ミリリットル中にポリリボソームを含む半透明のペレットを再懸濁し、上下にピペッティングしてよく混ぜます。勾配上にロードする前に30分間氷上サスペンション残りをしましょう​​。 </li></ol></li></ol> 5。ショ糖勾配超遠心分離に抽出ロード<ol><li>濃度を測定分光光度計を用いて、細胞質抽出物中に存在するRNAの。慎重にゆっくりと15パーセント〜55％ショ糖勾配上に〜抽出物の20 OD 260単位をロードします。利用可能なスペース2-3 mmは遠心分離中のチューブをオーバーフローを回避するために、管の表面に存在することを確認してください。 </li><li> 4℃で、TH-641超遠心ローターを用い23万×gで2時間30分の超遠心機を用いて勾配を遠心分離遠心分離が終了すると、チューブを外し、勾配を乱さないように注意深く氷の上に置きます。 </li></ol>ポリソームプロファイリングのための細胞質抽出物の6。画<ol><li>製造者によって記載されているように、自動化された密度分画システムにショ糖勾配を配置し、分画の自動分別と収集を進める（0.5ミリリットルずつ）。 </li><li> 254 nmの吸光度を連続的に監視して、個々のエッペンドルフチュー​​ブに各画分（〜500μl）を収集します。 parall中勾配分別操作のELは、polyribosomalプロファイルはチャート紙で編集されます。代わりに、ポリソームプロファイルの電子買収を持つようにチャートレコーダーに接続され、データ取得手段を使用しています。 </li><li>各実行の最後に、ドライアイス上で収集したエッペンドルフを転送します。このとき、ストアのいずれかを-80℃で画分を集め、以下のように直接またはタンパク質-RNA複合体を沈殿させる。 </li></ol> 7。タンパク質抽出および分析<ol><li>ショ糖勾配の各収集画分に、100パーセントの冷エタノール2容量を追加して、RNA-タンパク質複合体は、-20℃で一晩沈殿させてみましょう。 </li><li>遠心機で4℃で20分間11,000 xgで各RNA-タンパク質沈殿物を70％エタノールで洗浄する。乾燥した特異的抗体を用いたウェスタンブロットに進む前に、SDS-PAGEサンプル緩衝液中に沈殿物を再懸濁する（ 図2参照）。 </li></ol> 8。RNA抽出およびAnalysis <ol><li>セクション7.1のように、RNA-タンパク質複合体を沈殿させる。 0.1％SDSを含有する溶解緩衝液中の各RNA-タンパク質沈殿物を再懸濁する。 55℃の水浴中で30分間、2 mg / mlのプロテ​​イナーゼKを有する沈殿物のタンパク質成分を消化する。 </li><li> &quot;：クロロホルムフェノール」、クロロホルム2ボリューム、2 MのNaOAc pHが4の0.1ボリュームと20分間、4℃で万×gで遠心分離の1ボリュームを追加することにより、沈殿物のRNA成分を抽出。 1容量のイソプロパノールおよびグリコーゲン0.2μgの/μLを添加することにより、-20℃で一晩、各試料の得られた水相から沈殿物のRNA。 4℃で10,000×で1時間沈殿物を回転70％冷エタノールでRNAペレットを洗浄します。 </li><li> RNA分解酵素を含まない水を少量にRNAペレットを再懸濁します。量を評価 と品質 RNAを分光光度計を用いて。 </li></ol>

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著者らは、開示することは何もありません。

スクロース勾配上のポリソームプロファイル解析は、細胞または組織から単離したポリソーム9,11-14の濃度を分析することによって、翻訳開始の測定を可能にする。この手法は、 生体内で翻訳開始を測定するのに最適な（ない場合は一意の）アプローチである。これは、細胞周期15の間、細胞を増殖させるの並進状態を監視するために、及び翻訳開始には、ウイルス感?...

真核細胞は、常に迅速な適応細胞応答を必要とする有害な生理学的および環境ストレス条件の範囲に遭遇する。細胞ストレス応答は、抗生存と生存促進作用性因子との間の正確なバランスを必要とする。このバランスを崩壊させることは、癌や神経変性疾患などのヒトの病理の発展をリードする不可逆的な結果をもたらす可能性がある。ストレス反応の第一段階の間、細胞は、mRNAの翻訳のレベルでの遺伝子発現の変化の協調制御を伴う生存促進経路を活性化する。 真核生物のmRNAの翻訳は、翻訳開始因子（EIFS）、特異的なRNA結合タンパク質（のRBD）と、RNA分子の1との間の協調的な相互作用を伴う複雑な細胞プロセスである。開始、伸長、および終結：mRNA翻訳、3つの異なるフェーズに分割される。すべての3つのフェーズがレギュレーションの対象となりますがlatory機構は、翻訳制御メカニズムは、主にこのようにしてタンパク質合成2の律速段階を構成する翻訳の開始位相を、標的とする。 翻訳開始は 、 私は開始前複合体の形成につながる、三元複合体と40Sリボソームサブユニットへのその後の結合に会った eIF2a.GTP.Met-tRNAの形成に始まる高度に秩序プロセスです。次のステップは、そのようなeIF4F複合eIF3と、翻訳開始因子の活性が関与するmRNAに前開始複合体の動員である。このように形成された48S開始前複合体は、開始コドンAUGを認識するまで、mRNAの5 &#39;非翻訳領域のスキャンを開始するには、この機械を有効に特定の立体構造変化を受ける。翻訳開始因子の多くは、その後解放され、60Sサブユニットが翻訳のために、複雑な有能なリボソーム80Sを形成するために動員され、Aトンその時点でタンパク質合成を開始する（ 図1）。一染色体は、いわゆるポリソーム（またはポリリボソーム）の製造時に同じmRNAを翻訳することができ、複数の80S。ポリソームmRNA上の密度は、開始、伸長および終結率を反映し、したがって、特定の転写物の翻訳可能性の尺度である。しかしながら、ポリソームプロファイルは、主に開始段階におけるmRNA翻訳の変化を評価するために使用される。ここでは、翻訳開始阻害剤としてのプロテアソーム阻害剤を使用している。この薬物による癌細胞の処理は、翻訳開始因子eIF2a をリン酸化し、3 HRIというストレスキナーゼの活性化によって特徴付けストレス応答を誘導する。 eIF2aのリン酸化は、哺乳動物細胞4における翻訳開始の阻害を引き起こす主要なイベントの一つです。

<table border="0" cellpadding="0" cellspacing="0" width="875">
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	<tbody>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
			<td height="21" style="height:21px;width:279px;">Cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">HeLa cervical cancer cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Schneider Drosophila embryonic cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CRL-1963</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Schneider&#8217;s Drosophila Medium</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>SO146-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM</td>
			<td>Life technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">FBS</td>
			<td>Fisher Scientist Scientist</td>
			<td>SH30396-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">penicillin/streptomycin</td>
			<td>Life technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Sucrose solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">D-Sucrose</td>
			<td>Fisher Scientist</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Blue Bromophenol</td>
			<td>Fisher Scientist</td>
			<td>B3925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tris Hydrochloride</td>
			<td>Fisher Scientist</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:279px;">MgCl2 </td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NaCl</td>
			<td>Tekniscience</td>
			<td>3624-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DTT</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D 9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nonidet P40 (Igepal CA-630 )</td>
			<td>MJS Biolynx</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SDS</td>
			<td>Tekniscience</td>
			<td>4095-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td>
			<td>Life technologies</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Antiproteases (complete, mini, EDTA free)</td>
			<td>Roche</td>
			<td>11,836,170,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">RNA Extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteinase K</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenol: Chloroforme</td>
			<td>Fisher Scientist</td>
			<td style="width:119px;">BP1754I-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Chloroforme</td>
			<td style="width:311px;">Fisher Scientist</td>
			<td style="width:119px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycogen</td>
			<td>Life technologies</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isopropanol</td>
			<td style="width:311px;">Acros organics</td>
			<td>327270010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-FMRP antibody</td>
			<td colspan="2" style="width:429px;">Fournier et al., Cancer Cell International, 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-Ribosomal Protein L28 antibody</td>
			<td style="width:311px;">Santa Cruz Biotechnology, Inc.</td>
			<td style="width:119px;">SC-50362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Others</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteasome inhibitor : Bortezomib</td>
			<td style="width:311px;">LC Laboratories</td>
			<td style="width:119px;">B-1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DEPC (Diethylpyrocarbonate)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D5758-25ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RNaseZAP Solution</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">T25 cell culture flask</td>
			<td style="width:311px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1cc U100 Insulin Syringe 28 G1/2</td>
			<td>Fisher Scientist</td>
			<td>148291B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube ultra-centrifugation, PA, 12ml</td>
			<td>Fisher Scientist</td>
			<td>FSSP9763205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco Model 160 gradient former</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Sorvall OTD Combi</td>
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Thermo Scientific Sorvall Rotor TH-641</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;">54295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Automated Density Fractionation System</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;">67-9000-177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco UA-6 UV-vis detector</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ultracentrifuge C 5415</td>
			<td style="width:311px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Optical Microscope</td>
			<td style="width:311px;">Olympus CK2</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation initiation during stress conditions by polysome profiling

mRNA翻訳の正確な制御は、特に、生理学的および病理学的ストレスに応答して、真核細胞の恒常性に必須である。このプログラムの変更は、損傷した細胞の増殖、癌発生の顕著な特徴、または例えば神経変性疾患において見られるような時期尚早の細胞死につながることができる。翻訳調節の分子基盤について知られていることの多くは、密度勾配分画システムを用いてポリソーム分析から得られた。この手法は、線形スクロース勾配上で細胞質抽出物の超遠心分離に依存しています。回転が完了すると、システムは、異なる翻訳に対応し、遠心分離ゾーンの分画および定量化は、このようにポリソームプロファイルをもたらす、リボソーム集団を可能にする。ポリソームプロフィールの変化は、ストレスの種々のタイプに応答して生じる翻訳開始の変化や欠陥の指標である。この技術は、目を評価することができ電子翻訳開始の特定のタンパク質の役割、および特定のmRNAの翻訳活性を測定する。ここでは、正常または応力のいずれかの培養条件で、真核細胞および組織の翻訳開始を評価するために、ポリソームプロファイルを実行するために我々のプロトコルが記載されている。

前述のように、ポリソームプロフィールはストレス条件下で翻訳開始の変化の分析を可能にする。 図1は、前述のように、翻訳開始の簡略図が翻訳開始複合体の順序付けられたアセンブリーを含む多段階プロセスである。正常な成長条件下で、翻訳開始複合体は、その検出ソームプロファイルによる活性翻訳開始のための証明（;未処理図2）ポリリボソームに変換さ?...

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

ここでは、ストレス条件に応答する真核細胞のmRNA翻訳の開始の変化を分析するための方法を記載している。このメソッドは、非翻訳リボソームからリボソームを翻訳するショ糖勾配に速度の分離に基づいています。

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

analysis-translation-initiation-during-stress-conditions-polysome

Research

JoVE Journal

Biology

18.2K ビュー.  Laval University. ここでは、ストレス条件に応答する真核細胞のmRNA翻訳の開始の変化を分析するための方法を記載している。このメソッドは、非翻訳リボソームからリボソームを翻訳するショ糖勾配に速度の分離に基づいています。 

ビデオ: Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

ストレス応答時には植物ホルモンのモニタリング

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

生物学

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

ルシフェラーゼ免疫系による抗体のプロファイリング（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ゼブラフィッシュ胚における酸化ストレスの分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

ゲノム規模で哺乳類Translatomesのソーム分画および分析

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

工場ポリソームプロファイリングのための簡単​​な方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP顕微鏡によるmiRNAのギャップジャンクション依存的転写の解析

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

イン・ヴィトロ E3ユビキチンリガーゼ関数の解析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...