この作品は、米国国立科学財団の助成金＃MCB-0741914とMCB-0843627によってサポートされていました、追加のインフラストラクチャのサポートは、健康の2国立研究所の研究資源のナショナルセンターからG12 RR03060-26でニューヨーク市立大学で提供されていました。我々は感謝してJKCにはM82（野生型）およびCM15（変異）トマトのキューティクルを提供するためのコーネル大学の植物生物学部門でグループをローズ認める。我々は、AFMの実験と彼の寛大な助けのために教授アレクサンダーCouzisのCCNY化学工学グループから博士スピロスMonastiriotisに感謝します。我々は、グラフィカルな設計をサポートするために氏ローレン郷原に感謝します。

<ol>
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A., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysical+design+of+plant+cuticles:+an+overview.">The biophysical design of plant cuticles: an overview.</a> New Phytologist. 189, 938-949 (2011).</li><li>Bargel, H., Koch, K., Cerman, Z., Neinhuis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+the+plant+cuticle+and+cuticular+waxes+-+a+smart+material?.">Structure-function relationships of the plant cuticle and cuticular waxes - a smart material?.</a> Functional Plant Biol. 33, 893-910 (2006).</li><li>Jetter, R., Sch&#228;ffer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Composition+of+the+Prunus+laurocerasus+Leaf+Surface.+Dynamic+Changes+of+the+Epicuticular+Wax+Film+during+Leaf+Development.">Chemical Composition of the Prunus laurocerasus Leaf Surface. Dynamic Changes of the Epicuticular Wax Film during Leaf Development.</a> Plant Physiol. 126, 1725-1737 (2001).</li><li>Vogg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+fruit+cuticular+waxes+and+their+effects+on+transpiration+barrier+properties:+functional+characterization+of+a+mutant+deficient+in+a+very-long-chain+fatty+acid+Î²-ketoacyl-CoA+synthase.">Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid Î²-ketoacyl-CoA synthase.</a> J. Exper. Botany. 55, 1401-1410 (2004).</li><li>Isaacson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutin+deficiency+in+the+tomato+fruit+cuticle+consistently+affects+resistance+to+microbial+infection+and+biomechanical+properties,+but+not+transpirational+water+loss.">Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss.</a> Plant J. 60, 363-377 (2009).</li><li>Round, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Water+on+the+Nanomechanical+Behavior+of+the+Plant+Biopolyester+Cutin+as+Studied+by+AFM+and+Solid-State+NMR.">The Influence of Water on the Nanomechanical Behavior of the Plant Biopolyester Cutin as Studied by AFM and Solid-State NMR.</a> Biophysical J. 79, 2761-2767 (2000).</li><li>Fung, B. M., Khitrin, A. K., Ermolaev, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Broadband+Decoupling+Sequence+for+Liquid+Crystals+and+Solids.">An Improved Broadband Decoupling Sequence for Liquid Crystals and Solids.</a> J. Magn. Reson. 142, 97-101 (2000).</li><li>Morcombe, C. R., Zilm, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+shift+referencing+in+MAS+solid+state+NMR.">Chemical shift referencing in MAS solid state NMR.</a> J. Magn. Reson. 162, 479-486 (2003).</li><li>Fang, S. J., Haplepete, S., Chen, W., Helms, C. R., Edwards, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+atomic+force+microscopy+images+using+spectral+methods.">Analyzing atomic force microscopy images using spectral methods.</a> J. App. Phys. 82, 5891-5898 (1997).</li><li>Pollard, M., Beisson, F., Li, Y., Ohlrogge, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+lipid+barriers:+biosynthesis+of+cutin+and+suberin.">Building lipid barriers: biosynthesis of cutin and suberin.</a> Trends. Plant Sci. 13, 236-246 (2008).</li><li>Stark, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+studies+of+structure+and+dynamics+in+fruit+cuticle+polyesters.">NMR studies of structure and dynamics in fruit cuticle polyesters.</a> Solid State Nucl. Mag. 16, 37-45 (2000).</li><li>Schaefer, J., Stejskal, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon-13+nuclear+magnetic+resonance+of+polymers+spinning+at+the+magic+angle.">Carbon-13 nuclear magnetic resonance of polymers spinning at the magic angle.</a> J. Amer. Chem. Soc. 98, 1031-1032 (1976).</li><li>Sachleben, J. R., Chefetz, B., Deshmukh, A., Hatcher, P. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+relaxation+studies+of+plant+polyester+dynamics.+2.+Suberized+potato+cell+wall.">Nuclear magnetic resonance relaxation studies of plant polyester dynamics. 2. Suberized potato cell wall.</a> Macromolecules. 25, 149-154 (1992).</li><li>Bennett, A. E., Rienstra, C. M., Auger, M., Lakshmi, K. V., Griffin, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteronuclear+Decoupling+in+Rotating+Solids.">Heteronuclear Decoupling in Rotating Solids.</a> J. Chem. Phys. 103, 6951-6958 (1995).</li><li>Metz, G., Wu, X. L., Smith, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ramped-Amplitude+Cross-Polarization+in+Magic-Angle-Spinning+NMR.">Ramped-Amplitude Cross-Polarization in Magic-Angle-Spinning NMR.</a> J. Magn. Reson. Ser. A. 110, 219-227 (1994).</li><li>Peersen, O. B., Wu, X. L., Smith, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+CP-MAS+Signals+by+Variable-Amplitude+Cross-Polarization+-+Compensation+for+Inhomogeneous+B-1+Fields.">Enhancement of CP-MAS Signals by Variable-Amplitude Cross-Polarization - Compensation for Inhomogeneous B-1 Fields.</a> J. Magn. Reson. Ser. A. 106, 127-131 (1994).</li><li>lessandrini, A., Facci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+a+versatile+tool+in+biophysics.">AFM: a versatile tool in biophysics.</a> Measurement Sci. &#38; Tech. 16, 10-1088 (2005).</li><li>Ben&#237;tez, J. J., Matas, A. J., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+plant+biopolyester+cutin+by+AFM+and+spectroscopic+techniques.">Molecular characterization of the plant biopolyester cutin by AFM and spectroscopic techniques.</a> J. Struct. Biol. 147, 179-184 (2004).</li><li>Koch, K., Neinhuis, C., Ensikat, H. J., Barthlott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+assembly+of+epicuticular+waxes+on+living+plant+surfaces+imaged+by+atomic+force+microscopy+(AFM).">Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM).</a> J. Exper. Bot. 55, 711-718 (2004).</li><li>Muller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+A+Nanotool+in+Membrane+Biology.">AFM: A Nanotool in Membrane Biology.</a> Biochemistry. 47, 7986-7998 (2008).</li><li>Last, J. A., Russell, P., Nealey, P. F., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Applications+of+Atomic+Force+Microscopy+to+Vision+Science.">The Applications of Atomic Force Microscopy to Vision Science.</a> Investigative Ophthalmology &#38; Visual Science. 51, 6083-6094 (2010).</li></ol>

利害の衝突が宣言されません。

プロトコルは、ここに破壊的な化学分解を必要とせずに複雑な難治性の植物材料の詳細な分子とミクロ特性評価を可能に説明します。我々は、異種クチクラブレンドからepicuticularとintracuticularワックスの選択的な除去のための手順を実施し、監視対象のクチクラアセンブリの構造組織を制御するさまざまな脂質（ワックス）とクチンのバイオポリエステルのブレンドは、10を検討する。固体13 C NMRは、栽培、野生型および単一遺伝子からcutinsの架橋機能を比較するためにワックス分子成分の抽出を判断するために使用され、原子間力顕微鏡は、表面粗さの付随的な変化を調べるのに役立った。6,11ました変異体トマト果実、固体13 C NMRは、CHOおよびCH 2 Oの化学成分の相対的な数値を推定するために使用されていました。 設計機能の数このプロトコルのsは注目に値する。ワックス材料が発散極性の溶剤の一連の果実表皮の治療、脂質の広い範囲を包含するように徹底的な脱脂を達成するために不可欠です。さらに、脱蝋時間は8時間からキューティクルのサンプルの性質に応じて24時間ごとに異なる​​可能性があります。無傷の果実の表皮から一貫しepicuticularワックスを抽出するためには、表面に均一に接着剤コーティングを適用することが不可欠です。 ソリッドステートCPMAS 13 C NMR 12は 、そのネイティブの物理的特性を維持しながら、高度に不均一と不溶性の植物の生体高分子の様々な構造要素を識別するための迅速定性法である、13の伝統的な溶液状態のNMRも抽出ワックス混合物を特徴付けるために使用することができます。官能基の定量は、無傷の植物ポリマー、5高忠実度の直接偏波マジック角回転（Dのために望まれている場合PMAS）13 C NMR 5,14は、相補的な方法として使用する必要があります。官能基の正確な定量には、リサイクル回数、励起パルスの長さ、および異種デカップリングの強さの慎重な最適化を必要とする15異種デカップリングTPPM 16またはを使用して、50 kHzから185 kHzまでの範囲の1 Hの電界強度を設定することができ脊髄7の方法。これらのパラメータに加えて、CPMAS測定の感度はスピンロックの時間とハートマン·ハーン一致条件に依存しています。伝統的なCPMASの代わりに15が 、傾斜振幅CP（RAMP-CP）技術は、クロスを最大化するために実装することができますスピンロック期間（またはその逆）の間に一定の13 Cの電界強度の振幅を維持しながら直線的に（〜20から50パーセント）または接線方向に1 Hの振幅を変化させることにより、偏光効率でCPMAS測定を行う17,18二つの異なるROの最小Torの紡糸の周波数スペクトルのメインピークからスピニングサイドバンドを区別することが不可欠です。 コンタクトモードで行わ同時AFM測定には、ろうの成分の逐次除去時にインスタンスの高いスキャン速度と高解像度、19とキューティクルの表面状態の直接的なイメージングを可能にします。横方向（剪断）力による損傷を回避し、試料表面の削り、繊細な&quot;ソフト&quot;の植物材料の表面特性評価のための代替として使用することができます（非接触）モードをタップでAFMを動作させる。いずれの場合において5,20 、表面上の同じ場所の複数の画像の連続取得は、AFM測定の&quot;プローブ·表面相互作用&quot;に起因するどのような表面の損傷を識別するのに役立ちます。6,21最適な再現性については、ソフトクチクラ表面に適したバネ定数を有するAFMプローブがなければなりません使用され、温度と湿度の恒常性が維持されるべきである。6,15,20 </sまで固体NMRのに対し&gt;はアンサンブル平均（バルク）トマト果実キューティクルのプロパティの分子プロファイルを提供しており、原子間力イメージングは、これらの絶妙な複雑な巨大分子集合体の表面形状を追跡するための補完的な非侵襲性のプローブ22,23を提供しています。1,2

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog no.</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pectinase</td>
			<td>TCI America</td>
			<td>P0026</td>
			<td>EC 3.2.1.15; 10 U ml-1, store in refrigerator</td>
		</tr>
		<tr>
			<td>Cellulase</td>
			<td>Sigma-Aldrich</td>
			<td>C1184-100KU</td>
			<td>EC232.734.4; 1.3 units/mg, store in refrigerator</td>
		</tr>
		<tr>
			<td>Glacial Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-100G</td>
			<td>Extremely hazardous</td>
		</tr>
		<tr>
			<td>Incubator/shaker</td>
			<td>New Brunswick Scientific Co.</td>
			<td>Model No.G24</td>
			<td>MFG No.M1036-000G</td>
		</tr>
		<tr>
			<td>Vacuum Oven</td>
			<td>Precision Scientific</td>
			<td>31566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Variac Controller</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sintered glass thimble (85 mm/25mm)</td>
			<td>VWR</td>
			<td>89056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable extraction thimble ( 80 mm/ 25 mm)</td>
			<td>VWR</td>
			<td>28320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>EMD-MX0485-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>VWR</td>
			<td>RK20789</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>VWR</td>
			<td>82027-452</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>EM-CX1050-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>H302-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>VWR</td>
			<td>89002-984</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kim wipes</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma</td>
			<td>G9752</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.6 mm fastMAS zirconia rotor</td>
			<td>Varian (Agilent)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NMR spectrometer</td>
			<td>Varian 600 NMRS</td>
			<td>standard bore magnet</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>49419</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Adamantane</td>
			<td>Sigma-Aldrich</td>
			<td>100277</td>
			<td>To calibrate 90&deg; pulse in NMR</td>
		</tr>
		<tr>
			<td>Multimode Scanning Probe Microscope (Nanoscope IIIA)</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>Version 5.30r3sr3 (2005)</td>
		</tr>
		<tr>
			<td>AFM probe (Nonconductive silicon nitride tip)</td>
			<td>Veeco (Bruker AXS)</td>
			<td>Model NP-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnetic puck</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>VWR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fruit Peeler</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>VWR</td>
			<td>89038</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and biophysical study of fruit cuticles

キューティクル、陸生植物の空中部分に疎水性の保護層は、様々な生物と非生物的ストレスへの多彩な防御壁として機能し、また外部環境からの水の流れを調節します。1バイオポリエステル（クチン）と長鎖脂肪酸（ワックス）がキューティクルの主要な構造的な枠組みを形成する;。クチクラ層の機能の整合性は、外側の&#39;epicuticular&#39;層だけでなく、クチンの生体高分子と&quot;intracuticular &#39;ワックスから成るブレンドに依存して2ここで、我々は包括的なプロトコルを記述する商業的トマト（Solanum lycopersicum）の果実キューティクルから徹底的にワックスを抽出したり、キューティクルのコンポジットから順次選択してepicuticularとintracuticularワックスを除去する。ジェッターとシェーファー（2001）の方法は、果実の表皮からepicuticularとintracuticularワックスの段階的な抽出のために適応されました。3,4監視するにはシーケンシャルワックス除去、ソリッド·ステート·交差偏波マジック角スピニングのプロセス（CPMAS）13 C NMR分光法についての情報を補完するバルク材料の分子レベルの構造のプロファイルを提供し、原子間力顕微鏡（AFM）と並列に使用されましたマイクロスケールの地形とクチクラ表面の粗さ。栽培、野生型および単一遺伝子変異体トマト果実からの脱蝋キューティクルの架橋能力を評価するには、MAS 13 C NMRは酸素族（CHOおよびCH 2 O）の化学成分の相対比率を比較するために使用されていました。 様々な極性の溶剤のパネルで段階的にソックスレー抽出による徹底的な脱蝋は、クチンのバイオポリエステルの化学構造を維持しながら、彼らの脂肪族および芳香族成分の疎水性の特性に基づいて、ワックス部分を分離するための効果的な手段を提供します。 epicuticularワックス、セレの機械的な抽出補完的な物理的方法論によって監視intracuticularワックスのctive除去、キューティクルアセンブリを調査するための前例のない手段を提供します。このアプローチでは、超分子組織とワックスの様々なタイプの構造的統合、クチンワックスマトリックスのアーキテクチャ、および化学物質を明らかに各成分の組成物。さらに、固体13 C NMRは、野生型と変異型赤熟したトマト果実のCHOおよびCH 2 Oの化学成分の相対的な数値の違いを明らかにする。 NMR技術は、指紋に、不溶性アモルファス、化学的に不均一であるクチクラ物質の分子構造を優れたツールを提供しています。非侵襲性の面選択的イメージング技術として、AFMは、NM-μmの長さスケールのクチクラアセンブリの構造組織を調べるために、効果的かつ直接的な手段を供給する。

Watch this Scientific Journal Video about Isolation and Biophysical Study of Fruit Cuticles at JoVE.com

Isolation and Biophysical Study of Fruit Cuticles

空中植物器官は表皮、超分子バイオポリエステルワックスアセンブリによって保護されています。我々は固体NMRと原子間力顕微鏡による分子とミクロスケールのトマト果実キューティクルからの選択的に除去するエピとintracuticularワックス、それぞれを監視するために、と設計されたクチクラbiopolyestersの架橋能力を評価するためのプロトコルを提示します。

果実キューティクルの単離と生物物理学的研究

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

isolation-and-biophysical-study-of-fruit-cuticles

Research

JoVE Journal

Biology

16.5K ビュー.  City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies. 空中植物器官は表皮、超分子バイオポリエステルワックスアセンブリによって保護されています。我々は固体NMRと原子間力顕微鏡による分子とミクロスケールのトマト果実キューティクルからの選択的に除去するエピとintracuticularワックス、それぞれを監視するために、と設計されたクチクラbiopolyestersの架橋能力を評価するためのプロトコルを提示しま?...

ビデオ: Isolation and Biophysical Study of Fruit Cuticles

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

ショウジョウバエの勉強攻撃（ミバエ類）

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

コンピュータによって生成された動物モデルの刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

の単離と精製ショウジョウバエ</em磁気ビーズソート別>末梢ニューロン

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

電子鼻を使用した果実の揮発性の分析

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

ゲノムDNAから5  - ヒドロキシの選択的捕獲

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

細菌分裂機械の超解像イメージング

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代謝率の測定に<em&gt;ショウジョウバエ</em&gt;呼吸計測を使用した

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

べん毛モーターの機能の生物物理学的特性評価

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...