المؤلف روبرت Holdcraft بفضل المساعدة التقنية. وقد تم تمويل هذه الدراسة جزئيا من منحة وزارة الزراعة الأميركية الخاصة CSREES (2009-34155-19957) والصناديق يفقس (NJ08192).

<ol>
	<li>Dicke, M., Van Loon, J. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multitrophic+effects+of+herbivore-induced+plant+volatiles+in+an+evolutionary+context.">Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context.</a> Entomol. Exp. Appl. 97, 237-237 (2000).</li><li>Mumm, R., Dicke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+natural+plant+products+and+the+attraction+of+bodyguards+involved+in+indirect+plant+defense.">Variation in natural plant products and the attraction of bodyguards involved in indirect plant defense.</a> Can. J. Zool. 88, 628-628 (2010).</li><li>Hopke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore-induced+volatiles:+the+emission+of+acyclic+homoterpenes+from+leaves+of+Phaseolus+lunatus+and+Zea+mays+can+be+triggered+by+a+&#946;-glucosidase+and+jasmonic+acid.">Herbivore-induced volatiles: the emission of acyclic homoterpenes from leaves of Phaseolus lunatus and Zea mays can be triggered by a &#946;-glucosidase and jasmonic acid.</a> FEBS Lett. 352, 146-146 (1994).</li><li>Rodriguez-Saona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+the+emerald+ash+borer,+Agrilus+planipennis,+to+induced+volatiles+of+Manchurian+ash,+Fraxinus+mandshurica.">Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica.</a> Chemoecology. 16, 75-75 (2006).</li><li>Rodriguez-Saona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore+induced+volatiles+in+the+perennial+shrub,+Vaccinium+corymbosum,+and+their+role+in+inter-branch+signaling.">Herbivore induced volatiles in the perennial shrub, Vaccinium corymbosum, and their role in inter-branch signaling.</a> J. Chem. Ecol. 35, 163-163 (2009).</li><li>Karban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage-induced+resistance+in+sagebrush:+volatiles+are+key+to+intra-+and+interplant+communication.">Damage-induced resistance in sagebrush: volatiles are key to intra- and interplant communication.</a> Ecology. 87, 922-922 (2006).</li><li>Frost, 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=Within-plant+signalling+by+volatiles+overcomes+vascular+constraints+on+systemic+signalling+and+primes+responses+against+herbivores.">Within-plant signalling by volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores.</a> Ecol. Lett. 10, 490-490 (2007).</li><li>Heil, M., Bueno Silva, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-plant+signaling+by+volatiles+leads+to+induction+and+priming+of+an+indirect+plant+defense+in+nature.">Within-plant signaling by volatiles leads to induction and priming of an indirect plant defense in nature.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 5467-5467 (2007).</li><li>Turlings, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploitation+of+herbivore-induced+plant+odors+by+host-seeking+parasitic+wasps.">Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps.</a> Science. 250, 1251-1251 (1990).</li><li>Makovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatiles+involved+in+the+nonhost+rejection+of+Fraxinus+pennsylvanica+by+Lymantria+dispar+larvae.">Volatiles involved in the nonhost rejection of Fraxinus pennsylvanica by Lymantria dispar larvae.</a> J. Agric. Food Chem. 44, 929-929 (1996).</li></ol>

ليس لدي ما يكشف

ووصف دفع سحب جهاز جمع متقلبة هنا يمثل الطريقة القياسية للمجموعات فراغ الرأس من المواد المتطايرة النبات. وقد استخدم هذا الجهاز لتحديد استجابة متقلبة للأوراق التوت إلى الحيوانات العاشبة من يرقات عثة الغجر ، وسمح لي أيضا أن تقدم أدلة جديدة لدور HIPVs في داخل مصنع الإشارا?...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> غرف جمع متقلبة </td><td> التحليلية بحوث النظم ، وشركة </td><td> VCC - G6X12DT - 1P </td><td> جينزفيل ، فلوريدا </td></tr><tr><td> ضاغط الهواء ، و 20 غالونا ، والنفط مجانا ، 2 حصان </td><td> غربا </td><td> 3JR71 </td><td> غرينجر المباعة من قبل ، وشركة </td></tr><tr><td> تسليم نظام الهواء </td><td> التحليلية بحوث النظم ، وشركة </td><td> VCS - ADS - 4AFM4C </td><td> جينزفيل ، فلوريدا </td></tr><tr><td> نظام جمع الهواء </td><td> التحليلية بحوث النظم ، وشركة </td><td> VCS - MVCS - 4CX1P </td><td> جينزفيل ، فلوريدا </td></tr><tr><td> مضخة الفراغ 100 150V ، ربع حصان </td><td> غاست التصنيع ، وشركة </td><td> <a href="http://www.grainger.com/Grainger/GAST-Vacuum-Pump-4F740">4F740</a> </td><td> غرينجر المباعة من قبل ، وشركة </td></tr><tr><td> الميثيل jasmonate </td><td> سيغما الدريخ &lt;/ TD&gt; <td> J2500 </td><td> سانت لويس ، MO </td></tr><tr><td> توين - 20 </td><td> سيغما الدريخ </td><td> 93773 </td><td> سانت لويس ، MO </td></tr><tr><td> رودامين - B </td><td> سيغما الدريخ </td><td></td><td> سانت لويس ، MO </td></tr><tr><td> زجاجات البلاستيك الرش ، 2 أوقية </td><td> شركة Setco </td><td></td><td> Cranbury ، NJ </td></tr><tr><td> الأكمام البوليستر نسج </td><td> روكينجهام الفرص كورب </td><td></td><td> Reidsville ، NC </td></tr><tr><td> س فائقة الفخاخ جمع متقلبة </td><td> التحليلية بحوث النظم ، وشركة </td><td> VCT-1/4X3-SPQ </td><td> جينزفيل ، فلوريدا </td></tr><tr><td> سليل صورة برامج </td><td> سليل المؤسسة </td><td></td><td> فريدريك ، دكتوراه في الطب </td></tr><tr><td> ثنائي كلورو ميثان </td><td> سيغما الدريخ </td><td> 270997 </td><td> سانت لويس ، MO </td></tr><tr><td> الغاز اللوني HP 6890 </td><td> هيوليت باكارد </td><td></td><td></td></tr><tr><td> الغاز اللوني فاريان 3400 </td><td> فاريان </td><tد&gt; </td><td></td></tr><tr><td> ن اوكتان </td><td> سيغما الدريخ </td><td> 296988 </td><td> سانت لويس ، MO </td></tr><tr><td> مطياف الكتلة MAT 8230 </td><td> Finnigan </td><td></td><td> سان خوسيه ، كاليفورنيا </td></tr><tr><td> HP - 1 العمود GC </td><td> اجيلنت تكنولوجيز </td><td></td><td> بالو ألتو ، CA </td></tr><tr><td> MDN GC - 5S العمود </td><td> Supelco ، وشركة </td><td></td><td> Bellefonte ، والسلطة الفلسطينية </td></tr></table>

herbivore-induced blueberry volatiles and intra-plant signaling

وتنبعث عادة المتطايرة الناجمة عن مصنع عاشب (HIPVs) من النباتات بعد هجوم 1،2 عاشب. وتنظم هذه HIPVs أساسا من حمض محطة دفاعية jasmonic هرمون (JA) والمتذبذب jasmonate الميثيل المشتقة (ميجا) 3،4،5. على مدى العقود الماضية الباحثين 3 وقد وثقت أن HIPVs يمكن صد أو جذب الحيوانات العاشبة ، وجذب الأعداء الطبيعية من الحيوانات العاشبة ، وفي بعض الحالات التي يمكن أن تحدث أو دفاعات النبات الوزراء قبل هجوم عاشب. في ورقة حديثة 6 ، ذكرت أن التغذية من يرقات عثة الغجر ، تطبيق ميخيا الخارجية ، والأضرار الميكانيكية حمل الانبعاثات من المواد المتطايرة من النباتات عنبية ، ولو بشكل مختلف. بالإضافة إلى ذلك ، عنبية فروع الاستجابة لHIPVs المنبعثة من الفروع المجاورة للمحطة نفسها من خلال زيادة مستويات JA ومقاومة العواشب (أي دفاعات النبات مباشرة) ، وانبعاثات فتيلة متقلبة (أي دفاعات النبات غير المباشرة). العثور مماثلةوقد أبلغ مؤخرا عن الميرمية بالجلسات 7 ، 8 الحور ، والفاصوليا 9.. هنا ، وأنا أصف طريقة دفع سحب لجمع المواد المتطايرة الناجمة عن عنبية عاشب (عثة الغجر) التغذية ، تطبيق ميخيا الخارجية ، والأضرار الميكانيكية. وحدة جمع متقلبة يتكون من غرفة ل 4 جمع متقلبة ، على مقصلة 2 قطعة ، ونظام لتنقية الهواء إيصال الهواء واردة ، ونظام فراغ متصلة فخ مليئة سوبر س الممتزات لجمع المواد المتطايرة 5،6،10 . وeluted المتطايرة التي تم جمعها في سوبر س الفخاخ مع ثنائي كلورو ميثان وانفصلوا ثم كميا باستخدام غاز الكروماتوغرافي (GC). وقد استخدم هذا الأسلوب لجمع متقلبة ن 6 دراستي للتحقيق في استجابة متقلبة من الفروع التالفة التعرض لالمتطايرة من فروع عاشب المتضررة داخل النباتات عنبية. يتم وصف هذه الأساليب هنا. باختصار ، يتعرض لفروع عنبية التالفة HIPVs جيئة وذهابام الفروع المجاورة في نفس النبات. باستخدام التقنيات نفسها التي وصفها أعلاه ، يتم جمع المواد المتطايرة المنبعثة من الفروع بعد التعرض لHIPVs وتحليلها.

Watch this Scientific Journal Video about Herbivore-induced Blueberry Volatiles and Intra-plant Signaling at JoVE.com

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

يوصف أسلوب دفع سحب لجمع المواد المتطايرة النبات. الأسلوب يسمح للمقارنة بين المواد المتطايرة الناجمة عن التغذية عاشب ، jasmonate الخارجية الميثيل ، والأضرار الميكانيكية. كما تستخدم هذه التقنية للتحقيق في استجابة تقلبا من الفروع التالفة التعرض لالمتطايرة من فروع عاشب المتضررة داخل النباتات عنبية.

عاشب متطايرة بفعل العنبية واشارة الى داخل المصنع

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

herbivore-induced-blueberry-volatiles-and-intra-plant-signaling

Research

JoVE Journal

Biology

16.3K Views.  Rutgers University. يوصف أسلوب دفع سحب لجمع المواد المتطايرة النبات. الأسلوب يسمح للمقارنة بين المواد المتطايرة الناجمة عن التغذية عاشب ، jasmonate الخارجية الميثيل ، والأضرار الميكانيكية. كما تستخدم هذه التقنية للتحقيق في استجابة تقلبا من الفروع التالفة التعرض لالمتطايرة من فروع عا?...

Video: Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

تقنيات التصوير كاليفورنيا 2 + في اشارة الى الحيوانات المنوية البشرية

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

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.

البارافين ، جزءا لا يتجزأ من والمجمدة أقسام ذبابة الفاكهة</emالعضلات الكبار>

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

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

الاستقطاب إزفاء من البروتينات في نيون القيطم الأدمة في الاستجابة لاشارة Wnt

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

تصور كا الأوعية الدموية 2 + اشارة سببها ريوس المشتقة نظير الصماوي

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

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.

Electroantennographic الأحيائي كأداة فحص لمواد متطايرة النبات المضيف

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

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

تحفيز الميكانيكية التي يسببها الكالسيوم انتشار الموجات في الخلية monolayers: مثال من الأبقار الخلايا البطانية القرنية

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

الأيضية وصفها وغشاء التقطير للالمقارن تحليل البروتين من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الثقافات تعليق خلية

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

العزلة وثقافة الكبار ماوس العضلية للاشارة الخليوي و<em&gt; في المختبر</em&gt; تضخم القلب

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

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.

قياس معدل الأيض في<em&gt; ذبابة الفاكهة</em&gt; باستخدام قياس التنفس

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

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED الحرارية تدفق - الجمع بين علم البصريات الوراثي مع التدفق الخلوي

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...