Duennwald実験室での作業は、老化研究（遠く）、遺伝病財団（HDF）、ウィリアム·ウッド財団のためのアメリカ連合からの助成金によってサポートされています。

1。酵母における有毒PolyQ膨張タンパク質の発現体系的な分析は、酵母7で毒性を生成するために必要とされるpolyQ膨張タンパク質の正確なアミノ酸配列を確立しています。この有毒polyQ膨張タンパク質は、図を参照して、ハンチンチンタンパク質、polyQ領域、およびカルボキシ末端融合の元の配列からの蛍光タンパク質（GFPまたはCFPのいずれかに17個のアミノ酸に続いて、アミノ末端FLAG-タグが含まれています。 1）。誘導性と相対的な強力なGAL1プロモーター7、9の制御下で発現した場合46グルタミン以上のpolyQ膨張を有するタンパク質の発現は、（72と103グルタミンなど）酵母の毒性を生成します。 前述のように、酵母でpolyQの毒性は、そのプリオンの立体構造のタンパク質Rnq1pを運ぶ細胞でのみ明らかである、[RNQ +]例えば、酵母菌株W303 9。毒性polyQ膨張蛋白質RecapitulatesなどpolyQ長さに依存した毒性（下記参照）と集約（図1 b）のように酵母でpolyQ - 生物学の中心的な側面を、。特に、毒性polyQ膨張タンパク質は、すぐに酵母細胞を死滅させず、むしろ損なうまたは細胞周期や細胞ディビジョンを逮捕することにより、プレートまたは液体培養（未発表データ）上に酵母のコロニーの成長を減速または阻害する。 2。酵母細胞の潜在的な問題は有毒PolyQ膨張タンパク質を発現するそうでなければ毒性polyQ膨張タンパク質を発現する酵母細胞は、任意の成長欠陥9を表示しない場合があります。 polyQ毒性のこれらの抑制の遺伝的性質は、単純なメンデルの変異に基づいてされていないようですので、比較的高い周波数（我々の未発表の結果）で発生することができます。我々は、これらの自発的な抑制は、同様に[RNQ +] polyQ toxicity.Thesを決定する際に機能する正体不明のプリオンの硬化によって引き起こされると推測しているpolyQ毒性の電子自発的な抑制は、polyQ毒性を特徴付けるために、またはpolyQ毒性の修飾子を識別し、特徴づけることを目指して、任意の実験の成功を危うくすることができます。 これらの自発的な抑制の頻発を避けるために、非常に効果的であることが実証されている以下に示す予防策を、次のとおりです。 <ol><li>毒性実験のために新鮮な酵母細胞を使用しています。長時間酵母を保管しないでください。頻繁に凍結ストックから、新鮮な酵母のコロニーを取得します。 </li><li>頻繁に蛍光顕微鏡で毒性polyQ膨張タンパク質の発現および集計を監視します。 </li><li>任意の毒性の測定を開始する前にすべての回で毒性polyQ毒性（唯一の炭素源としてグルコースを含有する選択培地でIE）の発現を抑制する培地中で酵母細胞を保持します。 </li><li>各polyQ毒性実験のために少なくとも3つの独立した形質転換体を使用しています。&lt;/ LI&gt; </ol> 3。成長アッセイ<ol><li>各実験条件については、唯一の炭素源としてグルコースと酵母の選択培地3 mlの毒性polyQ膨張タンパク質を保有する酵母細胞の3つの独立した形質転換体から各コロニーを接種する。 </li><li> 30℃で一晩これらの培養物をインキュベート℃に培地中のグルコースは、それらの発現を抑制するので、これらの条件下で、細胞がpolyQ膨張タンパク質を発現されていません。これらの培養物の生い茂るさせてはいけません。1以下の一晩培養物のOD 600（600 nmの波長の光の吸光度）を保持します。 </li><li>我々は日常的に有毒なpolyQ膨張タンパク質を発現する酵母細胞の増殖の欠陥を監視するための3つの異なるアッセイを使用します。 </li></ol>プレート上で3.1成長<ol><li>私は選択的に0.0005のOD 600（すなわち、OD600 = 0.5文化の1:1000希釈）に一晩酵母培養（220 rpmの振盪しながら成長した）で希釈グルコースを含む直径7。 </li><li>均等に選択的な唯一の炭素源としてグルコースを含む培地と、唯一の炭素源としてガラクトースを含有する選択培地で1皿で1皿（直径10cm）にそれぞれ希釈した培養液50μlを（CAが得プレート当たり700コロニー）に広がる。 </li><li> 30℃で3〜4日のためにプレートをインキュベートインキュベーション後、各プレートの写真を撮ると、グルコースとガラクトースプレート上のコロニー数のコロニー数をカウントします。非常に有毒polyQ膨張タンパク質（103Q、図2a）を発現する酵母細胞を使用する場合は、理想的な条件下では、ガラクトースプレート上にないか、ごく少数のコロニーがあってはならない。 </li></ol> 3.2スポッティングアッセイこのアッセイは、メッキアッセイは、上記よりも定量的であり、したがって、同じプレート上で同じ実験とpolyQ毒性でも微妙な違いを明らかにすることができます。 <ol><li> overnを希釈IGHT文化は、0.1のOD 600にグルコースを含む培地で増殖させた。 </li><li>滅菌96ウェルプレートに、これらの希釈培養の200μlのピペットおよびマルチチャンネルピペットを用いて滅菌水で5つの五倍希釈系列を調製する。 </li><li> 、フロッガーを使用して（また、細胞懸濁液を転送するための8×6ピンで、スポッターと呼ばれる）を唯一の炭素源と唯一の炭素源としてガラクトースと選択培地を含むプレートとしてグルコースと選択培地を含むプレートに細胞懸濁液を転送します。 </li><li>プレートは三から四日間30℃で、それらをインキュベートする前に乾燥することができます。 </li><li>インキュベーション後、各プレートの写真（図2b）を取る。 </li></ol> 3.3。液体培養物の増殖によって、酵母のpolyQ毒性を監視するこのプロトコルは、ここで説明した3つのアッセイ（OD600番号）が最も定量的であるとさえpolyQ毒性の非常に微妙な違いを検出することができます。前述のOC自発的抑制のcurrenceしかし、潜在的に誤解を招くような結果を生むことができます。従って私は上述した二つのメッキアッセイの少なくともいずれかで、このアッセイを組み合わせることをお勧めします。我々は、これらの実験のためにBioscreenCインストゥルメントを使用することを提案する。 BioscreenCは、定義された撹拌を使用して定義された温度で培養しながら、自動的に100ウェルプレートで酵母培養液の光学密度を測定する計器です。酵母細胞の成長とその光学密度を測定するための他の方法も適用される場合があります。 <ol><li>唯一の炭素源として3 mlの滅菌水で3回グルコースを含む最少培地から酵母細胞を洗浄します。 </li><li> 0.1のOD 600にガラクトースを含む培地で増殖させた一晩培養物を希釈します。 </li><li>酵母培養の300μlの100ウェルBioscreenCプレートの各ウェルを記入してください。 </li><li>簡単Bioscreen実験プログラムを開きます。あなたは（空白と培地のみを含む監視するサンプル数を決定するコントロール）は、30℃に温度を設定し、3日に実験の長さを設定、15分に測定間隔を設定し、600 nmのフィルター/ブラウン設定し、で、各測定の前に15秒の揺れのモードを設定中強度。 </li><li> BioscreenC楽器と付属のソフトウェアは、実験中に撮影し、各データポイントのExcelスプレッドシートを生成します。 </li><li>各サンプルのExcelでの成長曲線を準備し、別のサンプル（図2c）の成長を比較します。 BioscreenC実験によって生成されたデータの分析の詳細な説明は、10の前に提供されています。 </li></ol> 4。代表的な結果<img alt="図1" src="/files/ftp_upload/3461/3461fig1.jpg" /> 図1。酵母polyQモデル。短い、非毒性polyQを発現する酵母細胞を示す毒性polyQ膨張タンパク質bのa）の模式図）蛍光顕微鏡拡張タンパク質（25Q、左パネル）および酵母細胞は長い間、毒性polyQ膨張タンパク質（103Q、右のパネル）を発現する。 <img alt="図2" src="/files/ftp_upload/3461/3461fig2.jpg" /> 図2。 polyQ膨張タンパク質を発現する酵母細胞の増殖アッセイの代表的な結果。 1）めっきアッセイ約700酵母細胞がプレート上に広げ、30℃で三日間℃でインキュベートした。上部パネルは、グルコース培地を含むプレートを示し、毒性polyQ膨張タンパク質の発現（103Q）が誘導されない 、すなわち。下のパネルには、ガラクトースを含む培地酵母プレートを示し、毒性polyQ膨張タンパク質の発現が誘導される、すなわち。 B）スポッティングアッセイは、ここに示されている実験では、全く自発的な抑制が発生していないことに注意してください。いずれかの非毒性polyQタンパク質（25Q）または毒性polyQ膨張proteiを保有する酵母細胞の5つのシリアル5倍希釈では、N（103Q）は、タンパク質の発現を抑制するグルコースプレート（左パネル）またはそれらの発現を誘導し、ガラクトースプレート上にスポットした。次いで、プレート℃のC）BioscreenC実験を行った。どちらに非毒性polyQタンパク質（25Q）または毒性polyQ膨張タンパク質を発現する酵母細胞の培養物の成長が（103Q）によって監視された30℃で3日間インキュベートした。 BioscreenC楽器。実験条件とBioscreenC実験の解析は、メインテキストに記載されています。

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この原稿は有毒polyQ膨張タンパク質を発現する酵母細胞の減少成長に基づいて、モデル生物の酵母でpolyQ毒性を測定する3つの相補的な実験的アプローチについて概説します。酵母での作業は、polyQ膨張タンパク質9,11,12の折り畳みおよび毒性を含む、タンパク質のミスフォールディングとその後の毒性の基本的な細胞および分子メカニズムに深い洞察を提供してきました。ここで提示さ...

<table border="1"><tbody><tr><td> 楽器の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>フロッガー（6x8ピン） </td><td> V＆Pサイエンティフィック、サンディエゴ</td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurvesアメリカ</td><td> 5101370 </td></tr><tr><td> 100よくハニカムプレート</td><td> Growthcurvesアメリカ</td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

タンパク質の折り畳みは、アルツハイマー病、パーキンソン病、ハンチントン病1など多くのヒトの疾患、特に神経変性疾患、関連付けられています。ハンチントン病（HD）は、タンパク質ハンチンチン内のポリグルタミン（polyQ）領域の異常な拡大によって引き起こされます。 polyQ -拡張ハンチンチンタンパク質が異常なコンフォメーション（つまりmisfolds）を達成し、細胞毒性2を発生します。少なくとも8つの更なる神経変性疾患は、脊髄小脳性運動失調とケネディ病3を含むpolyQ -拡張によって引き起こされる。 モデル生物の酵母の影響内および分子間polyQ毒性の要因とpolyQ膨張を発現する細胞で損なわれている細胞経路の識別を含む、polyQ毒性の細胞および分子基盤に重要な洞察を容易にしましたタンパク質3-8。重要なLYは、酵母で発見されたpolyQ毒性の多くの側面は、このようにpolyQ毒性を支える基本的なメカニズムの発見のための酵母モデルの意義を実証し、他の実験システムやHD患者からのサンプルである程度再現された。 酵母でpolyQ毒性を決定するために直接、比較的簡単な方法は、polyQ膨張タンパク質を発現する酵母細胞の増殖の欠陥を測定することです。本稿ではpolyQ膨張タンパク質を発現する酵母細胞の増殖を測定することにより、酵母でpolyQ毒性を決定するために3つの相補的な実験的アプローチを説明しています。最初の二つの実験的アプローチは、プレート上で酵母の増殖を監視し、第三のアプローチはBioscreenC楽器を用いた液体酵母培養物の増殖を監視します。 さらに、この原稿は、酵母polyQモデルを扱うときに発生する可能性のある実験的な難しさを説明し、または回避するのに役立ちます戦略を概説しこれらの問題を最小限に抑えることができます。ここで説明したプロトコルが識別するために、遺伝的経路とpolyQ毒性を調節する小分子を特徴付けるために使用することができます。また、記載のアッセイは、酵母のモデル内の他の疾患に関連したミスフォールドタンパク質によって引き起こされる毒性の正確な分析のためのテンプレートとして機能する可能性があります。

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

本稿ではポリグルタミンの毒性を評価するための3つの相補的なプロトコル（polyQ）拡張酵母のタンパク質について説明します。<em&gt;サッカロマイセスセレビシエ</em&gt;。これらのプロトコルは、簡単に酵母内の他のミスフォールドタンパク質の毒性を監視するために変更することができます。

成長アッセイ酵母におけるポリグルタミン毒性を評価する

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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13.5K ビュー.  Boston Biomedical Research Institute. 本稿ではポリグルタミンの毒性を評価するための3つの相補的なプロトコル（polyQ）拡張酵母のタンパク質について説明します。サッカロマイセスセレビシエ。これらのプロトコルは、簡単に酵母内の他のミスフォールドタンパク質の毒性を監視するために変更することができます。

ビデオ: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

出芽酵母の複製寿命の測定

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

生物学

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

酵母の菌タイプIIIエフェクターの発現により誘導される増殖阻害表現型の同定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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

甲殻類との長期致死毒性試験アルテミアfranciscana</em

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

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

筋肉の細胞内コンパートメントを評価するための方法<em&gt; C。エレガンス</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

SPRi、ナノのSPRiおよびELISAアッセイを用いて血清中のヒト成長ホルモンの比較分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY：酵母増殖の多パラメータ定量化のための大規模法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

酵母におけるポリグルタミン毒性の試金を使用して融合パートナーの蛍光タンパク質の効果

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2 +の効果の評価のためのスクリーニング アッセイ中スループット-シグナリングおよびひと精子先体反応

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...