This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

注：脾臓をマウス（6〜10週齢）に由来し、すべての動物実験は、ピーター·マッカラム癌センター（E486）の動物倫理ガイドラインに従って行った。 サンプルの調製。 <ol><li>活性化したマウスNK細胞の調製。 <ol><li>市販のキットを用いたネガティブ選択により（GZMB遺伝子ヌル）マウス- / - C57BL / 6またはB6.GrzmBの脾臓の単一細胞懸濁液からの一次ナイーブなNK細胞を単離する。 </li><li>メーカーのプロトコルに従ってください。簡単に説明すると、磁気的TおよびB細胞、樹状細胞、顆粒球、マクロファージ、およびそれらの特定の表面マーカーに対するビオチン標識抗体を用いて赤血球などの「非NK」細胞を標識する。 「非NK」細胞を枯渇させる抗ビオチンビーズと磁気分離を使用してください。 </li><li>培養物を24ウェルプレートに70万細胞/ mlのIL-2（1,000 U / ml）を含む培地中で5~7日間、NK細胞を単離する37時&#160; °C。 </li><li>あるいは、使用は19 T細胞、OT1 T細胞受容体トランスジェニックマウス20からの一次抗原制限CTLは、CTL分泌GZMB 19の活性を決定するために、活性化NK細胞からの混合リンパ球培養21、または上清中に生成された活性化した。 </li></ol></li><li>ヒトNK細胞株および一次NK細胞の単離の維持。 <ol><li> 10％ウシ胎児血清を補充したロズウェルパーク記念研究所（RPMI）培地中のヒトNK白血病（YT細胞）を維持する。代替的に、健康な被験者の末梢血から一次NK細胞を精製し、以前に22を説明したように100 U / mlのIL-2を含む培地中で2~3日間刺激する。 </li></ol></li><li>細胞溶解物を生成する。 <ol><li> PBS（pH7.4）中で細胞を3回洗浄し、次いで、ノニデットP-40溶解緩衝液に懸濁（0.1％NP-40、250のNaCl、25mMのヘペス、2.5mMのEDTA）。理想的には、5×10 6の最小値を溶解</sNK細胞、または100μlの緩衝液中の1×10 7 T細胞&gt;アップ。多くはセリンプロテアーゼの不可逆的阻害剤であるように、プロテアーゼ阻害剤を追加して、そのようなGZMAなどの特定のトリプターゼでしないでください。 20分間氷上でインキュベートし、次いで10分間15000×gで微量で核をペレット化。核ペレットを廃棄し、新しいチューブに上清を移す。 </li></ol></li><li>溶解物のタンパク質濃度を決定します。 <ol><li>そのようなブラッドフォードやビシンコニン酸（BCA）、などの選択肢の市販のプロトコルを使用して、タンパク質濃度を決定する。濃度が〜2 mg / mlの最小であることを確認してください。必要になるまで-20℃で保存溶解物（グランザイム活動は-20℃で何ヶ月も安定している）。 </li></ol></li></ol> 2.グランザイムB活性アッセイ<ol><li>試薬の調製<ol><li> -20＆＃でアリコートに10 mMのDMSO（〜5 mg / mlの）における基板の株式（のBoc-AAD-S-BzlのまたはAC-IEPD-PNA）とストアを準備する176; C。たての作業希釈液を調製及びBoc-AAD-S-Bzlのは、部分的に、DMSO中のストレージに加水分解するとして、それぞれの日の後に捨てる。 </li><li> -20℃で250 mMのDMSO中の比色試薬（5,5&#39;-ジチオ - ビス（2-ニトロ安息香酸）、DTNB）の株式（0.09グラム/ ml）およびストアを準備します。毎日新鮮な作業希釈を準備します。この試薬は、のみSBzlインジケータグループ、しないのpNAを有する基板に必要です。 </li><li>新鮮な緩衝液（0.1M HEPES、0.05 MのMgCl 2、pH7.3）で2〜3週間毎を構成している。 </li></ol></li><li>室温にDTNBと合成基質を持参してください。基質（1：16、 例えばバッファの1.6ミリリットルで100μl）を希釈し、DTNB（1：250、 例えば 2.5ミリリットルバッファ内の10μl）を、よく混ぜる。 </li><li> 160μlの最終容量までバッファ内の各サンプルの100μgのタンパク質溶解物の最低希釈する。マルチチャンネルピペットを使用すると、 &#39;&#39; U &#39;&#39;底のビニール96ウェルプレートに（〜33.3 /ウェル）三重にウェルあたり50μlを添加する。（また、三重で）「バッファ専用」コントロールを含めます。 </li><li>のAc-IEPD-PNAを扱う際のサンプル/バッファ制御に希釈しDTNB100μlのを追加します（この手順を省略します。 注：GZMBにより交流-IEPD-pNAの切断は、直接蛍光性PNA（ 図4B）をリリースします。 </li><li>その後緩衝液を用いて任意の負の制御を開始し、期待陽性サンプルを終え、すぐに三重の各セットに希釈した基質50μlのを追加するためにマルチチャンネルピペットを使用してください。 </li><li>プレートリーダーでプレートを置きます。 OD 405 nmでマイクロプレートリーダーでのBoc-ALA-ALA-Aspを-S-Bzlの/ AC-IEPD-pNAの加水分解によるのAspアーゼ活性を測定する。 25の測定値、15秒毎（約6分の合計読み取り時間）を取る、運動アッセイプロトコルを使用してください。 </li><li>プレートリーダーのソフトウェアがVmaxの値を生成できない場合、手動で基質切断の速度を計算するために、各時点で生成された吸光度測定値を使用する。 </li></ol>ダ3.分析TA 注：生成されたデータは、時間（MOD /分）を超えるODの変化として定義された最大速度として提示される。 <ol><li>データを分析するために、吸光度の変化率から計算される基質切断の最大速度を決定する。通常は、このオプションを持って、プレートリーダーソフトウェアを使用して、これを自動的に行う。 </li><li>代わりに手動で、プレートリーダー上で動的アッセイ中に生成される吸光度の変化の速度論的プロットを、表示した後に最も急な直線を与えるデータポイントを選択します。ほとんどのアッセイの最大レートは、アッセイの最初の2分以内に達成される。 </li><li>時間間隔によって、この直線と除算で最高OD読み取りから初期ODの読みを引く。統計分析およびグラフィック表示のためのMicrosoft Excel /グラフパッドに生データを転送します。 </li></ol>

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The authors have nothing to disclose.

歴史的にグランザイムは、標的細胞の急速なアポトーシス死を誘導することができる細胞傷害性リンパ球（CTLおよびNK細胞）の重要なエフェクター分子として同定された。これは主に、アスパラギン酸（D）残基で標的基質分子を切断し、したがって両方の切断プロカスパーゼによってカスパーゼカスケードを活性化することができるだけでなく、それらの下流の標的のいくつかあったGZMBの作...

グランザイムは、ナチュラルキラー（NK）細胞および細胞傷害性Tリンパ球（CTL）1の分泌リソソームに見出されるセリンプロテアーゼのファミリーである。五つの異なるグランザイムは、ヒト（A、B、H、KとM）で存在し、そしてマウスにおける10（A - G、K、M及びN）2,3。グランザイムAおよびグランザイムB（GZMA、GZMB）が最も豊富であり、広範囲に人間と齧歯類の​​設定で研究されてきた。 GZMBの古典的な機能は、標的細胞の細胞質ゾル4にアクセスするためのグランザイムを可能にする孔形成タンパク質、パーフォリンと組み合わせて実行される、標的細胞におけるアポトーシスの誘導である。 GZMB発現が明確に細胞傷害性リンパ球において見出されているが、最近の研究では、好塩基球6、肥満細胞7、形質細胞様樹状細胞8、およびB細胞9を含むが、ケラチノサイト5に限定されるものではなく、他のGZMBを発現する種々の細胞型に対処されてきた、 10。この文脈では、非アポトーシスGZMB機能は炎症プロセス、組織リモデリングおよび他の免疫調節特性11-14の参加の範囲が明らかになった。 より広範な生物学的役割は、以前に疑われるよりもGZMBのために提案されていることを考えると、研究者は、その検出のための信頼性の高い、特定のツールが必要です。有利なのアスパラギン残基のカルボキシル側で切断するGZMBの特定の要件、真核生物のセリンプロテアーゼ15の中でユニークな特性である。マウス、ヒトおよびラットGZMBしかしマウスGZMBの拡張された基質特異性端子（P1）のAspを有する特定のジェネリック基質は、GZMBによって効率的に切断することができることを意味し、ヒトおよびラット16の両方とは微妙に異なるが、構造的に非常に類似しているすべての3種から、上流のP1のより複雑な配列をもつ他の基材に対し、広く多様な結果を与える可能性があります。過去と最近の李両方でterature、この事実は、注意深く制御、動態試験状況17を修正しようとしているにもかかわらず、いくつかの実験結果の生物学的意義のかなりの混乱や誤解を引き起こした。 本論文では、2つの市販の基板、すなわちのBoc-ALA-ALA-Aspを-SBzlとN-アセチルイルのGlu-Proの-ASP-p-ニトロアニリドを使用して、これらのポイントを説明しようとしてきた。 2つの試薬は、切断（蛍光無料パラニトロアニ対遊離スルフヒドリル）以下の異なる反応基を生成行いますが、これはタンパク質分解的切断には影響何もありません。記載されているプロトコルは、非常に古いプロトコル18の近代的な適応ですが、また、そのようなGZMAやGzmHなど他グランザイム、の活性を検出するための方法論的な枠組みを提供しながら、研究者は、適切に異なるGZMB基板を使用するように助けるべきである。

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			<td style="width:241px;">Company</td>
			<td style="width:181px;">Catalogue number</td>
			<td style="width:427px;">Comment/description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boc-Ala-Ala-Asp-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMSB05</td>
			<td>Granzyme B substrate (mouse and human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ac-IEPD-pNA&#160;</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMPNA009</td>
			<td>Granzyme B substrate (only human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-a-CBZ-L-lysine-S-Bzl</td>
			<td>Sigma-Aldrich</td>
			<td>C3647</td>
			<td>Granzyme A substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suc-Phe-Leu-Phe-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SB025</td>
			<td>Granzyme H substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5,5&#8217;-dithio-bis(2-nitrobenzoic acid)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td>&#160;DTNB, Ellman&#8217;s Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit II mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-657</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
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			<td height="21" style="height:21px;"></td>
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			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biorad iMark</td>
			<td></td>
			<td>Biorad Microplate Manager Software Version MPM6.3</td>
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			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
			<td></td>
		</tr>
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a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

のBoc-AAD-S-Bzlの基板は、GZMBに特異的である セリンプロテアーゼGZMBは細胞傷害性リンパ球（CTLおよびNK細胞）の主成分であり、そのようなウイルス感染または形質転換細胞などの標的細胞に迅速なアポトーシス死を誘導するのに主に責任がある。これはまた、アスパラギン残基の後に切断するが、プロテアーゼの異なるクラス、システインプロテアーゼファミ...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

のBoc-ALA-ALA-Aspを-S-Bzlのの加水分解：具体的にグランザイムBタンパク質分解活性を測定する比色アッセイ

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Research

JoVE Journal

Chemistry

12.9K ビュー.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

ビデオ: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

定性的には生体試料中のRNAおよびDNAを区別するために迅速比色アッセイ

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

化学

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

DNAグリコシラーゼ活性の定常状態、プレ定常状態、およびシングルターンオーバー速度論的測定

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

プロ蛍光基質アナログを経由してPAD4活性の蛍光ベースの監視

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

塩化物イオンの動的電気化学測定

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

表面酸化と還元を経て液体金属の表面張力を操作する方法

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of Phosphorus(I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable low-valent-phosphorus-containing compound acts as a useful P+ transfer agent. Unlike traditional methods employed to access low-coordinate phosphorus species which usually require pyrophoric phosphorus-containing precursors (white phosphorus, Tris(trimethylsilyl)phosphine, etc.), or harsh reducing agents (alkali metals, potassium graphite, etc.), the current approach does not involve pyrophoric or explosive reagents and can be done on large scales (&#62;20 g) in excellent yields by undergraduates with basic air-free synthetic training. The bromide counter ion is readily exchanged with other anions such as tetraphenyl borate (described herein) using typical salt metathesis reagents to obtain materials with desired properties and reactivities. The versatility of this P+ transfer approach is exemplified by the reactions of these triphosphenium precursors with an N-heterocyclic carbene and an anionic bisphosphine, each of which readily displace the neutral bisphosphine to give an NHC-stabilized phosphorus(I) cation and a phosphorus(I) containing zwitterion, respectively.

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of PhosphorusI

The synthesis of a triphosphenium bromide salt is described and its use as a P+ transfer agent is outlined by reactions with an N-heterocyclic carbene and an anionic bisphosphine, yielding an NHC-stabilized P(I) cation and a P(I) containing zwitterion, respectively.

Triphosphenium臭化物塩の調製と反応性：リンの便利で安定したソース（I）

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

HDAC1 と HDAC6 hela 細胞活性の同時測定セルを用いたクロマト MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

液晶エラストマーのアクチュエータのマイクロの準備

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

A Two-Step Protocol for Umpolung Functionalization of Ketones Via Enolonium Species

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. Recently, we have developed a two-step strategy for ketone enolate umpolung that has enabled the development of methods for chlorination, azidation, and amination using azoles. In addition, we have developed C-C bond&#8211;forming arylation and allylation reactions. At the heart of these methods is the preparation of the intermediate and highly reactive enolonium species prior to addition of a reactive nucleophile. This strategy is thus reminiscent of the preparation and use of metal enolates in classical synthetic chemistry. This strategy allows the use of nucleophiles that would otherwise be incompatible with the strongly oxidizing hypervalent iodine reagents. In this paper we present a detailed protocol for chlorination, azidation, N-heteroarylation, arylation, and allylation. The products include motifs prevalent in medicinally active products. This article will greatly assist others in using these methods.

A two step one-pot protocol for the umpolung of ketone enolates to enolonium species and addition of a nucleophile to the &#945;-position is described. Nucleophiles include chloride, azide, azoles, allyl-silanes, and aromatic compounds.

Enolonium 種によるケトン類の極性転換を高機能化 2 段階のプロトコル

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

高コンテンツ イメージング解析によるロドプシン輸送アッセイ

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...