Name: the identification of sea lamprey pheromones using bioassay-guided fractionation
Uploaded: 2018-07-17T15:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

米国地質調査ハモンド湾生物ステーションの研究施設の利用、米魚類野生生物局と水産のスタッフと海カナダ海ヤツメウナギを提供するために感謝しますこの研究は、李維、柯李五大湖漁業委員会からの補助金によって支えられました。

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魚は、化学の世界はまだ識別される化合物の完全に住んでいます。バイオアッセイ誘導分別識別しサクラマス31、アジア象32、および海ヤツメウナギ33で観察など、多くの化学的相互作用を仲介する生理活性分子を特徴付ける重要な証明されています 34,35。バイオアッセイ誘導分別が正確...

フェロモンは、食料源を検索、検出捕食者および個体1の社会的な相互作用を仲介することでそれらを支援する個人によってリリースされた特定の化学信号です。昆虫のフェロモン ・ コミュニケーションはずっとよく勉強2;しかし、化学的同定と水生脊椎動物フェロモンの生物学的機能が検討されていないとして広く。Id の知識とフェロモンのリリースの機能は、絶滅危惧種3,4または制御害虫種5,6の回復を容易に適用できます。これらの技術のアプリケーションは、分離・生理活性フェロモン成分の同定を必要とします。
フェロモンの同定は、天然物化学の枝です。フェロモン研究の進歩は、フェロモン分子自身の性質上、部分的に制限されています。フェロモンは、不安定になり、少量でリリースされた、揮発性7,8または9水溶性化合物の微量を検出するだけいくつかのサンプリング手法が存在します。フェロモン的なアプローチには、1) 知られている化合物、2) メタボロミクス、および 3) バイオアッセイ誘導分別の対象となるスクリーニングが含まれます。知られている化合物の標的スクリーニングは、フェロモンとして機能するいると仮定の生理的過程の市販の代謝の副産物をテストします。研究者のみ知られており、利用可能な化合物をテストできますので、この方法は制限されています。ただし、それをもたらしました金魚の性ホルモンの成功した識別関数フェロモン10、11,12として。メタボロミクスは、潜在的な生物学的システム13内低分子代謝産物を区別する第 2 フェロモン同定アプローチです。2 つのグループ (すなわちアクティブと非アクティブのエキス) の代謝プロファイルの比較により、潜在的な代謝プロファイルの id 構造の解明から代謝物を精製し、生物活性は確認された14です。特定の混合物の複雑な配合の添加剤または相乗効果、代謝物は一緒にではなく、分数13のシリーズとしてと見なされるために、メタボロミクスで検出される可能性が高い。まだ、メタボロミクスの実装は、結果のデータは、新規構造の解明を促進しないので合成参照の可用性に依存します。
バイオアッセイ誘導の分別、2 つのフィールドにまたがる統合、反復的なアプローチ: 化学および生物学。このアプローチは、分離とアクティブなフェロモン化合物の同定をガイドするのに生理・行動生物検定の結果を使用します。粗野なエキスは、 (すなわち、分子サイズ、極性等)の化学的性質によって分別され、生物検定および/または電気 olfactogram (EOG) 録音でのテストです。生理活性成分をスクリーニング、分別および EOGs および/または生物検定の手順を繰り返ししています。分子量と合成される化合物のテンプレートを生成する構造情報提供分光法と分光学的方法により純粋な活性化合物の構造を明らかにします。バイオアッセイ誘導の分別には、多様な代謝と生合成の経路から予測される可能性が高いではないユニークな化学のスケルトンと潜在的新規フェロモンをもたらすことができます。
ここでは、分離し、男性の海ヤツメウナギ セックス フェロモン化合物の生物活性を評価するために使用するバイオアッセイ誘導分別のプロトコルについて述べる。海ヤツメウナギ (カワヤツメ属 marinus) は、これらの魚は大きく 3 つの段階から成る、さくの生活史を仲介する化学手掛かりの嗅覚の検出に依存するため、フェロモン ・ コミュニケーションを研究する理想的な脊椎動物のモデルです。幼虫、少年、大人。海ヤツメウナギ淡水ストリームの底に穴を掘る、抜本的な変態、湖または海洋大ホスト魚を寄生彼らに移行する少年に変換します。ホストの魚からデタッチ後大人は産卵ストリームに戻って移行, ストリーム居住者幼虫15,16,17,18,19 発表渡り鳥のフェロモンに導かれて.成熟した雄は産卵場に登る、仲間を集めるし、約一週間、断続的に産卵して死ぬ15,20の多成分性フェロモンをリリースします。海ヤツメウナギ フェロモンの同定は、フェロモン通信システムの変調がラウレンツィアーナ五大湖21の侵襲の海ヤツメウナギにオプションの中では重要です。

<table><tbody><tr><td>Premium standard wall borosilicate capillaries with filament&amp;nbsp;</td><td>Warner Instruments</td><td>G150F-4</td><td>recording and reference electrode (OD 1.5 mm, ID 0.86 mm)</td></tr><tr><td>Pipette puller instrument</td><td>Narishige</td><td>PC-10</td><td>pulls electrodes for EOGs</td></tr><tr><td>Diamond-tipped glass cutter</td><td>Generic</td><td></td><td>cut tip of electrodes for EOG</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150-4</td><td>odorant delivery tube for EOG</td></tr><tr><td>Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm</td><td>Warner Instruments</td><td>ESP-M15N</td><td>recording electrode holder</td></tr><tr><td>Reference electrode holder E Series with handle with&amp;nbsp; Ag/AgCl pellet&amp;nbsp; for glass capillary OD 1.5 mm</td><td>Warner Instruments</td><td>E45P-F15NH</td><td>reference electrode holder</td></tr><tr><td>1 mm pin</td><td>Warner Instruments</td><td>WC1-10</td><td>to bridge reference and recording electrode holders</td></tr><tr><td>2 mm pin</td><td>Warner Instruments</td><td>WC2-5</td><td>to bridge reference and recording electrode holders</td></tr><tr><td>Agar</td><td>Sigma</td><td>A1296</td><td>molten agar to fill electrodes</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma</td><td>P9333</td><td>3M KCl to fill electrodes and electrode holders</td></tr><tr><td>Micropipette microfil</td><td>World Precision Instruments</td><td>MF28G-5</td><td>to fill electrodes and electrode holders</td></tr><tr><td>L-Arginine</td><td>Sigma</td><td>A5006</td><td>positive control odorant for EOG</td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td></td></tr><tr><td>Water bath</td><td>Custom made</td><td>N/A</td><td>holds odorants for EOG</td></tr><tr><td>3-aminobenzoic acid ethyl ester (MS222)</td><td>Syndel USA</td><td>Tricaine1G</td><td>EOG anesthetic</td></tr><tr><td>Gallamine triethiodide</td><td>Sigma</td><td>G8134-5G</td><td>EOG paralytic</td></tr><tr><td>1 mL syringe</td><td>BD Biosciences</td><td>301025</td><td>to administer paralytic</td></tr><tr><td>Subcutaneous needle 26G 5/8</td><td>BD Biosciences</td><td>305115</td><td>to administer paralytic</td></tr><tr><td>Roller clamp</td><td>World Precision Instruments</td><td>14043-20</td><td>adjust flow rate of anesthic into lamprey&#039;s mouth</td></tr><tr><td>Sodium chloride (NaCl)</td><td>J.T. Baker</td><td>3624-05</td><td>for preparation of 0.9% saline</td></tr><tr><td>V-shaped plastic stand as specimen stage</td><td>Custom made</td><td>N/A</td><td>holds lamprey during EOG</td></tr><tr><td>Plastic trough</td><td>Custom made</td><td>N/A</td><td>holds V-shaped plastic stand during EOG</td></tr><tr><td>Scalpel Blades - #11</td><td>Fine Science Tools</td><td>10011-00</td><td>for EOG dissection</td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools</td><td>10003-12</td><td>for EOG dissection</td></tr><tr><td>Straight ultra fine forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>for EOG dissection, Dumont #5SF Forceps</td></tr><tr><td>Curved ultra fine forceps</td><td>Fine Science Tools</td><td>11370-42</td><td>for EOG dissection, Moria MC40B</td></tr><tr><td>Straight pring Scissors</td><td>Fine Science Tools</td><td>15003-08</td><td>for EOG dissection</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Discovery V8</td><td>for EOG dissection</td></tr><tr><td>Illuminator light</td><td>Zeiss</td><td>CL 1500 ECO</td><td>for EOG dissection</td></tr><tr><td>Plastic tubing</td><td>Generic</td><td></td><td>to connect re-circulating EOG setup and water baths</td></tr><tr><td>Odorant delivery tubing</td><td>Custom made</td><td>N/A</td><td></td></tr><tr><td>In line filter and gasket set</td><td>Lee Company</td><td>TCFA1201035A</td><td></td></tr><tr><td>Micromanipulators</td><td>Narishige</td><td>MM-3</td><td>to position electrodes and odorant delivery capillary tube</td></tr><tr><td>Magnetic holding devices</td><td>Kanetec</td><td>MB-K</td><td></td></tr><tr><td>Valve driver</td><td>Arduino</td><td>custom made</td><td>to control the opening of the valve for odor stimulation</td></tr><tr><td>Electromagnetic valve</td><td>Lee Company</td><td>LFAA1201618H</td><td>valve for odor stimulation</td></tr><tr><td>NeuroLog AC/DC amplifier</td><td>Digitimer Ltd.</td><td>NL106</td><td>to increase the amplitude of the elictrical signal</td></tr><tr><td>NeuroLog DC pre-amplifier with headstage</td><td>Digitimer Ltd.</td><td>NL102G</td><td>to increase the amplitude of the elictrical signal</td></tr><tr><td>Low-pass 60 Hz filter</td><td>Digitimer Ltd.</td><td>NL125</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices LLC</td><td>Axon Digidata 1440A</td><td></td></tr><tr><td>Dell computer (OptiPlex 745) running Axoscope data acquistion software</td><td>Molecular Devices LLC</td><td>AxoScope version 10.4</td><td></td></tr><tr><td>Faraday cage</td><td>Custom made</td><td>N/A</td><td>Electromagnetic noise shielding</td></tr><tr><td>Two-choice maze</td><td>Custom made</td><td>N/A</td><td>waterproofed marine grade plywood covered with plastic liner</td></tr><tr><td>Trash pump</td><td>Honda</td><td>WT30XK4A</td><td>fills maze with water from nearby river</td></tr><tr><td>Peristaltic pump with tubing</td><td>Cole Parmer</td><td>Masterflex 07557-00</td><td>to adminster odorants in maze</td></tr><tr><td>Inverter Generator</td><td>Honda</td><td>EU1000i</td><td>powers perstaltic pump</td></tr><tr><td>Release cage</td><td>Custom made</td><td>N/A</td><td>used to acclimate lamprey in the maze</td></tr><tr><td>Mesh</td><td>Generic</td><td></td><td>used to contain the dimensions of the maze and minimize water turbulance with mesh rollers</td></tr><tr><td>Buckets (5 gallon)</td><td>Generic</td><td></td><td>to mix odorants</td></tr><tr><td>Flow meter</td><td>Marsh-McBirney</td><td>Flo-Mate 2000</td><td>to measure discharge</td></tr><tr><td>XAD 7 HP resin</td><td>Dow chemical</td><td>37380-43-1</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Water bath</td><td>Yamato</td><td>BM 200</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Freeze dryer</td><td>Labconco</td><td>CentriVap Concentrator</td><td>for extraction of conditioned water</td></tr><tr><td>chloroform</td><td>Sigma</td><td>CX1050</td><td>for isolation of fraction pools</td></tr><tr><td>Silica gel 70-230 mesh</td><td>Sigma</td><td>112926-00-8</td><td>for isolation of fraction pools</td></tr><tr><td>Silica gel 230-400 mesh</td><td>Sigma</td><td>112926-00-8</td><td>for isolation of fraction pools</td></tr><tr><td>Pre-coated silica gel TLC plates</td><td>Sigma</td><td>99571</td><td>for isolation of fraction pools</td></tr><tr><td>anisaldehyde</td><td>Sigma</td><td>A88107</td><td>for isolation of fraction pools</td></tr><tr><td>Sephadex LH-20</td><td>GE Healthcare</td><td>17-0090-01</td><td>for isolation of fraction pools</td></tr><tr><td>Amberlite XAD 7 HP resin</td><td>Sigma</td><td>XAD7HP</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>4, 2.5L capacity glass columns</td><td>Ace Glass Inc.</td><td>5820</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>TQ-S TOF LC Mass spectrometer (or equivalent)</td><td>Waters Co.</td><td>N/A</td><td>for structure elucidation</td></tr><tr><td>Binary HPLC pump</td><td>Waters Co.</td><td>1525</td><td>for isolation of fraction pools/compounds</td></tr><tr><td>Agilent NMR spectrometer, 900MHz (or equivalent)</td><td>Agilent</td><td>N/A</td><td>for structure elucidation</td></tr><tr><td>Rotovap drying system</td><td>Buchi</td><td>RII</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>UV lamp (254 nm)</td><td>Spectronics Co.</td><td>ENF-240C</td><td>for thin layer chromatography&amp;nbsp;</td></tr></tbody></table>

the identification of sea lamprey pheromones using bioassay-guided fractionation

バイオアッセイ誘導の分別は、生理・行動生物検定の結果を使用して分離とアクティブなフェロモン化合物の同定をガイドする反復的なアプローチです。このメソッドは、幅広い動物種のフェロモンとしてその関数化学信号の正常性状に起因しました。海ヤツメウナギは行動または生理学的な応答を仲介するフェロモンを検出する嗅覚に頼る。私たちはこの魚の生物学の知識を使用して推定されるフェロモンの機能を仮定するし分離とアクティブなフェロモン成分の同定。クロマトグラフィーは、抽出、集中、およびエアコンの水から化合物の分離に使用されます。電気 olfactogram (EOG) 録音する分数は嗅覚応答を引き出すを決定するため行われています。2 つの選択肢迷路行動アッセイは、臭気の分数のいずれかがアクティブな行動もし、好みを誘発するかどうかを決定する使用されます。分光法と分光学的方法は、分子量や構造解明を支援する構造の情報を提供します。純粋な化合物の生物活性を EOG と行動実験で確認しました。迷路の中にみられる行動応答は最終的に自然のストリーム設定の機能を確認するためのフィールド設定で検証しなければなりません。これらの生物検定は、1) 分別プロセスをガイドし、2) 確認するデュアル役割と、分離されたコンポーネントの活性の定義します。ここでは、分別のバイオアッセイ誘導アプローチの有用性を例証する海ヤツメウナギ フェロモン同定の代表的な結果を報告します。海ヤツメウナギ フェロモンの同定は、フェロモン通信システムの変調が侵襲的海ヤツメウナギ ラウレンツィアーナ五大湖にオプションの中では特に重要です。このメソッドは、イチイの広範な配列の化学交信を特徴付けるし、水系の化学生態学に光を当てるに容易に適応できます。

バイオアッセイ誘導分別のプロトコルで説明されている手順をまとめた図を図 1に示します。プロトコルには、分離し、構造、嗅覚の効力 5 推定海ヤツメウナギ フェロモン (図 2) の行動の活性を評価する手順が含まれます。質量分析および NMR データ (図 3および図 4) を使用して...

Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

ここでは、分離し、構造、嗅力海ヤツメウナギの推定フェロモン化合物の行動応答を評価するためのプロトコルを提案する.

バイオアッセイ誘導分別を使用して海ヤツメウナギ フェロモンの同定

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

the-identification-sea-lamprey-pheromones-using-bioassay-guided

Research

JoVE Journal

Biochemistry

8.9K ビュー.  Michigan State University. ここでは、分離し、構造、嗅力海ヤツメウナギの推定フェロモン化合物の行動応答を評価するためのプロトコルを提案する.

ビデオ: The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

環境学

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

爬虫類から皮膚脂質の分離、定量、化学分離

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

カリネクテス・サピドゥスにおける神経ペプチドの多面的質量分析調査

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and ...

化学

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

生化学

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

<meta charset="utf8"></head><body>C. elegansにおける揮発性性フェロモン応答の抽出と定量化

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

<meta charset="utf8"></head><body>著者スポットライト:揮発性性フェロモンがオスの C.エレガンスの 行動に及ぼす影響の調査

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

行動学

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

宿主植物の揮発性物質のスクリーニングツールとしてElectroantennographicバイオアッセイ

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

生物学

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegansの走化性アッセイ

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

バイオアッセイ誘導分別を使用して海ヤツメウナギフェロモンの同定

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この動画の章

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

爬虫類から皮膚脂質の分離、定量、化学分離

カリネクテス・サピドゥスにおける神経ペプチドの多面的質量分析調査

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

宿主植物の揮発性物質のスクリーニングツールとしてElectroantennographicバイオアッセイ

C. elegansの走化性アッセイ

バイオアッセイ誘導分別を使用して海ヤツメウナギ フェロモンの同定

要約

さらに動画を探す

この動画の章

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

爬虫類から皮膚脂質の分離、定量、化学分離

カリネクテス・サピドゥスにおける神経ペプチドの多面的質量分析調査

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

Caenorhabditis elegansにおける揮発性性フェロモン抽出と化学吸引アッセイ

宿主植物の揮発性物質のスクリーニングツールとしてElectroantennographicバイオアッセイ

C. elegansの走化性アッセイ

バイオアッセイ誘導分別を使用して海ヤツメウナギフェロモンの同定