המחבר מודה רוברט Holdcraft לקבלת סיוע טכני. מחקר זה מומן בחלקו על ידי משרד החקלאות CSREES מיוחד גרנט (2009-34155-19957) וקרנות לבקוע (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>

אין לי מה לחשוף

שכיבות למשוך מנגנון גבייה נדיפים המתואר כאן מייצג שיטה סטנדרטית אוספים אמיץ נדיפים של הצמח. מנגנון זה שימש כדי לקבוע את התגובה נדיף של עלי אוכמניות על ידי זחלי עש herbivory צוענית מותר גם לי לספק ראיות חדשות על תפקיד HIPVs איתות צמח פנים. התוצאות המוצגות כאן מראות כי האכלה זחל, exogenously-להחיל MeJA, ופצעו מכני מוגבר פליטת נדיפים באתר של נזק, אם כי באופן שונה. תוצאות אלו מצביעות כי מסלול JA ממלא תפקיד מרכזי ב אינדוקציה של HIPVs ב אוכמניות. חשיפה HIPV לא לגרום פליטת נדיפים. ענפים עם זאת, HIPV החשופים היו טעונים לתגובה תנודתי עלה ל האכלה עש צועני. בתגובה HIPVs ידי גידול פליטת נדיפים בהיעדר אוכלי עשב עלול להיגרם אקולוגיים כמו גם cos פיזיולוגייםTS, בגלל HIPVs עשוי לספק מידע אמין על אויבים טבעיים של אוכלי עשב. במקום זאת, אסטרטגיה אדפטיבית יותר עשוי להיות עבור הצמחים הממשלה עצמם לתגובת תנודתי מוגבר לאחר חשיפה HIPVs. זה שיעור מוגבר של פליטת נדיפים מן HIPV חשוף עלים עשוי לשמש הגנה עקיפה על ידי מעדיפים למשוך את האויבים הטבעיים של אוכלי עשב או עשוי למלא תפקיד ביטחוני ישיר מאז נדיפים מספר הנגרם על ידי עש הצועני אוכמניות יכולה להיות השפעה על זחלים דוחה 12. לסיכום, התוצאות שלי מראות כי העלים מהענפים שנפגעו אוכמניות להעביר מידע דרך נדיפים כי למזער את הנזק האכלה חרקים פליטת נדיפים הממשלה העלים קבלת סניפים סמוכים. תוצאות אלו, בשילוב עם אלה על ידי אחרים מספקים ראיות מוצקות לכך HIPVs לשחק תפקיד איתות צמח התוך ו נוספת להדגים את רב תפקודי תפקידי HIPVs.

<table border="1" style=";text-align:right;direction:rtl"><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> אוסף לתאי נדיפות </td><td> מחקר אנליטי Systems, Inc </td><td> VCC-G6X12DT-1P </td><td> Gainesville, פלורידה </td></tr><tr><td> מדחס אוויר, גל 20, שמן חופשי, 2 כ&quot;ס </td><td> מערבה </td><td> 3JR71 </td><td> נמכר על ידי גריינג&#39;ר, Inc </td></tr><tr><td> מערכת אספקת האוויר </td><td> מחקר אנליטי Systems, Inc </td><td> VCS-ADS-4AFM4C </td><td> Gainesville, פלורידה </td></tr><tr><td> Air מערכת איסוף </td><td> מחקר אנליטי Systems, Inc </td><td> VCS-MVCS-4CX1P </td><td> Gainesville, פלורידה </td></tr><tr><td> משאבת ואקום 100-150V, ¼ כ&quot;ס </td><td> גסט ייצור, Inc </td><td> <a href="http://www.grainger.com/Grainger/GAST-Vacuum-Pump-4F740">4F740</a> </td><td> נמכר על ידי גריינג&#39;ר, Inc </td></tr><tr><td> מתיל jasmonate </td><td> סיגמא אולדריץ &lt;/ Td&gt; <td> J2500 </td><td> סנט לואיס, מיזורי </td></tr><tr><td> Tween-20 </td><td> Sigma-Aldrich </td><td> 93773 </td><td> סנט לואיס, מיזורי </td></tr><tr><td> Rhodamine-B </td><td> Sigma-Aldrich </td><td></td><td> סנט לואיס, מיזורי </td></tr><tr><td> תרסיס בקבוקי פלסטיק, 2 גרם </td><td> Setco בע&quot;מ </td><td></td><td> Cranbury, NJ </td></tr><tr><td> פוליאסטר שרוולים חוט </td><td> Rockingham הזדמנויות קורפ </td><td></td><td> Reidsville, NC </td></tr><tr><td> Super-Q אוסף מלכודות תנודתי </td><td> מחקר אנליטי Systems, Inc </td><td> VCT-1/4X3-SPQ </td><td> Gainesville, פלורידה </td></tr><tr><td> Scion תוכנה תמונה </td><td> Scion Corporation </td><td></td><td> פרדריק, MD </td></tr><tr><td> Dichloromethane </td><td> Sigma-Aldrich </td><td> 270997 </td><td> סנט לואיס, מיזורי </td></tr><tr><td> גז כרומטוגרף HP 6890 </td><td> Hewlett Packard </td><td></td><td></td></tr><tr><td> גז כרומטוגרף וריאן 3400 </td><td> וריאן </td><tד&gt; </td><td></td></tr><tr><td> n-אוקטן </td><td> Sigma-Aldrich </td><td> 296988 </td><td> סנט לואיס, מיזורי </td></tr><tr><td> ספקטרומטר מסה MAT 8230 </td><td> פיניגן </td><td></td><td> בסן חוזה, קליפורניה </td></tr><tr><td> HP-1-GC עמודה </td><td> Agilent Technologies </td><td></td><td> בפאלו אלטו, CA </td></tr><tr><td> MDN-5S GC עמודה </td><td> Supelco, Inc </td><td></td><td> Bellefonte, הרשות הפלסטינית </td></tr></table>

herbivore-induced blueberry volatiles and intra-plant signaling

אוכלי עשב הנגרמת נדיפים צמח (HIPVs) נפלטים בדרך כלל אחרי 1,2 לתקוף אוכלי עשב מצמחים. HIPVs אלו מוסדר בעיקר על ידי חומצה jasmonic הגנתי הורמון צמח (JA) ו jasmonate מתיל נגזרות נדיף שלה (MeJA) 3,4,5. במהלך 3 העשורים האחרונים חוקרים תיעדו כי HIPVs יכול לדחות או למשוך אוכלי עשב, למשוך את האויבים הטבעיים של אוכלי עשב, ובמקרים מסוימים הם יכולים לגרום או הגנות הממשלה צמח לפני המתקפה אוכלי עשב. במאמר האחרונות 6, אני דיווחו כי האכלה של עש צועני זחלים, אקסוגניים יישום MeJA, ונזק מכני לגרום פליטת נדיפים מצמחים אוכמניות, אם כי באופן שונה. בנוסף, סניפים אוכמניות להגיב HIPVs הנפלט סניפים סמוכים של אותו צמח על ידי הגדלת רמות של JA והתנגדות אוכלי עשב (כלומר, הגנות צמח ישיר), ועל ידי פליטת תחול נדיף (כלומר, הגנות צמח עקיף). בדומה לאתרings דווחו לאחרונה 7 לענה, צפצפה 8, שעועית לימה 9 .. הנה, אני מתאר שיטה שכיבות למשוך לאיסוף נדיפים אוכמניות המושרה על ידי אוכלי עשב (עש צועני) האכלה, אקסוגניים יישום MeJA, ונזק מכני. יחידת איסוף נדיף מורכב תא 4 L נדיפים אוסף, הגיליוטינה 2 חלקים, מערכת אספקת אוויר מטהר האוויר הנכנס, ומערכת ואקום מחובר מלכודת מלא Super-Q הסופח לאסוף נדיפים 5,6,10 . נדיפים שנאספו Super-Q הם מלכודות eluted עם dichloromethane והפריד ואז לכמת באמצעות גז כרומטוגרפיה (GC). שיטה זו שימשה אוסף נדיפים n המחקר שלי 6 לחקור את התגובה נדיפים ענפים ניזוק חשיפה נדיפים מענפי אוכלי עשב שניזוק בתוך צמחים אוכמניות. שיטות אלה מתוארים כאן. בקצרה, סניפים אוכמניות ניזוק נחשפים HIPVs הלוך ושובמ סניפים שכנות בתוך אותו צמח. באמצעות אותן טכניקות שתוארו לעיל, נדיפים הנפלטים סניפים לאחר חשיפה HIPVs נאספו ונותחו.

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

שיטה שכיבות למשוך לאיסוף נדיפים צמח מתואר. השיטה מאפשרת השוואה של נדיפים המושרה על ידי האכלה אוכלי עשב, jasmonate מתיל אקסוגניים, ונזק מכני. טכניקה זו משמשת גם כדי לחקור את התגובה נדיפים ענפים ניזוק חשיפה נדיפים מענפי אוכלי עשב שניזוק בתוך צמחים אוכמניות.

אוכלי עשב הנגרמת נדיפים אוכמניות צמח תוך איתות

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

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16.3K Views.  Rutgers University. שיטה שכיבות למשוך לאיסוף נדיפים צמח מתואר. השיטה מאפשרת השוואה של נדיפים המושרה על ידי האכלה אוכלי עשב, jasmonate מתיל אקסוגניים, ונזק מכני. טכניקה זו משמשת גם כדי לחקור את התגובה נדיפים ענפים ניזוק חשיפה נדיפים מענפי אוכלי עשב שניזוק בתוך צמחים אוכמניות.

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

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.

מדורים פרפין-Embedded קפואים של תסיסנית סקס שרירים

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

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

הערכת נכסים איתות של Ectodermal epithelia במהלך פיתוח Craniofacial

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

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.

טרנסלוקציה מקוטב של חלבוני ניאון ב Xenopus האאקטודרם בתגובה איתות 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.

ויזואליזציה של כלי דם Ca 2 + איתות מופעלות על ידי ROS נגזר אוטוקריני

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.

המבדק Electroantennographic ככלי מיון עבור נדיפים צמחים מארח

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.

הרבייה מכאנית מושרה גירוי סיד גל בmonolayers הסלולרי: דוגמה לתאי אנדותל קרני שור

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.

מטבולית תיוג וממברנה היפוך חלוקה לניתוח השוואתי של proteomic<em&gt; Thaliana ארבידופסיס</em&gt; תא תרבויות השעיה

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.

בידוד והתרבות של שריר לב מאוס למבוגרים לתא איתות ו<em&gt; במבחנה</em&gt; לב היפרטרופיה

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.

מדידה של קצב חילוף החומרים ב<em&gt; דרוזופילה</em&gt; באמצעות Respirometry

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 Thermo Flow - שילוב Optogenetics עם cytometry זרימה

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