המחברים מודים בתודה תמיכה כספית מתקציב המדינה של הסוכנות למחקר סלובנית ו ESOPE של הנציבות האירופית הפרויקט (QLK-2002-02003), הממומנים במסגרת תכנית המסגרת 5. המחברים מכירים את התרומה של פרופ &#39;. מרקו Snoj, פרופ. מאיה Cemazar, מאטג&#39; Merkac ו Rado Likon בהכנת הכתבה-Video זו.

הליך הטיפול electrochemotherapy מורכב הזרקת סמים מקומי או מערכתי בעקבות המסירה של פולסים חשמליים להחיל הגידול 1. מידע מפורט על טיפול מתפרסם נהלי עבודה סטנדרטיים (SOP) 2. טיפול אינדיקציות: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> סוג Tumour: קרצינומה של התאים הבזליים, וגרורות עורית של מלנומה ממאירה, סרטן השד, hypernephroma, סרקומה של קפוסי ואחרים. </li><li style=";text-align:right;direction:rtl"> מיקום Tumour: עורית נגיש גושים תת עורית הגידול על כל חלקי הגוף. </li><li style=";text-align:right;direction:rtl"> גודל Tumour יכול להיות מ 3 מ&quot;מ עד 3-5 ס&quot;מ קוטר. </li><li style=";text-align:right;direction:rtl"> מספר גושים, בין 1 ל עד 100. </li></ol> הליך הטיפול: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדמה מקומית או כללית, בהתאם למספר גושים של מטופלים ואת המיקום. </li><li style=";text-align:right;direction:rtl"> הבחירה והתרופות: ציספלטין או bleomycin. ציספלטין ו bleomycin ניתן להזריק intratumorally, bleomycin גם דרך הווריד במינון של 15,000 IU / m 2. המינון המוזרק ליישום intratumoural של bleomycin הוא 250-1000 IU / 3 ס&quot;מ של רקמת הגידול ועל ציספלטין 0.5 -1 מ&quot;ג / ס&quot;מ 3 של רקמת הגידול </li><li style=";text-align:right;direction:rtl"> מרווח הזמן בין הממשל התרופה ויישום של פולסים חשמליים; לאחר ההזרקה intratumoral של התרופה באופן מיידי, לאחר עירוי לוריד לאחר 8 דקות. עד 28 דקות. </li><li style=";text-align:right;direction:rtl"> גנרטור חשמלי הדופק עבור electrochemotherapy אמור לספק מרובע גל פולסים חשמליים של משרעת 1000 V או יותר, תדירות החזרה של הרץ 1-5 קילוהרץ. </li><li style=";text-align:right;direction:rtl"> החשמל הדופק פרמטרים: 8 פולסים של לדוגמא 1000 V במרחק 8 מ&quot;מ בין שתי אלקטרודות בצלחת, 1 הרץ או 5 kHz תדירות, משך הדופק 100 μs. </li><li style=";text-align:right;direction:rtl"> אם את הגידול כולו אי אפשר להכיל בין האלקטרודות, היישום של פולסים חשמליים יש לחזור על כך פעמים רבות על מנת לכסות את אזור הגידול כולו, כולל שולי בטיחות. </li><li style=";text-align:right;direction:rtl"> סוג אלקטרודה: אלקטרודות צלחת משמשים, גושים קטנים הגידול שטחי, אלקטרודות מחט הם לטיפול גושים seeded עמוק מתחת לעור. </li></ol> תכונות אחרות של הטיפול: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בדרך כלל טיפול אחד יעיל לחיסול גידולים גולה, אולם electrochemotherapy ניתן לחזור במרווחים 4 שבועות על רקמת הגידול שנותר גושים או גידולים אחרים. </li></ol> בשל חוסר רעילות מערכתית - כי מינונים נמוכים מאוד של bleomycin או ציספלטין יש צורך - חולים יכולים להיות מטופלים על בסיס מחוץ המטופל, והוא יכול לעזוב את בית החולים מיד לאחר הטיפול.

<ol>
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Cancer. 98, 388-398 (2008).</li><li>Mir, L. M., Belehradek, M., Domenge, C., Orlowski, S., Poddevin, B., Belehradek, J., Schwaab, G., Luboinski, B., Paoletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemotherapy,+a+new+antitumor+treatment:+first+clinical+trial.">Electrochemotherapy, a new antitumor treatment: first clinical trial.</a> C. R. Acad. Sci. III. 313, (1991).</li><li>Mir, L. M., Glass, L. F., Sersa, G., Teissie, J., Domenge, C., Miklavcic, D., Jaroszeski, M. J., Orlowski, S., Reintgen, D. S., Rudolf, Z., Belehradek, M., Gilbert, R., Rols, M. P., Belehradek, J., Bachaud, J. 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R., Gehl, J., Collins, C. G., Snoj, M., Billard, V., Geertsen, P. F., Larkin, J. O., Miklavcic, D., Pavlovic, I., Paulin-Kosir, S. M., Cemazar, M., Morsli, N., Soden, D. M., Rudolf, Z., Robert, C., O'Sullivan, G. C., Mir, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemotherapy+-+an+easy,+highly+effective+and+safe+treatment+of+cutaneous+and+subcutaneous+metastases:+results+of+ESOPE+(European+Standard+Operating+Procedures+of+Electrochemotherapy)+study.">Electrochemotherapy - an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study.</a> EJC Suppl. 4, 3-13 (2006).</li><li>Quaglino, P., Mortera, C., Osella-Abate, S., Barberis, M., Illengo, M., Rissone, M., Savoia, P., Bernengo, M. 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גידול של cytotoxicity bleomycin על ידי חשיפה של תאים electroporative פולסים חשמליים תוארה לראשונה על ידי Okino M ו-LM מיר 3,4. לאחר מכן electrochemotherapy הודגם להיות יעיל גם עבור 5 ציספלטין. נתונים פרה נרחב נאספו על במבחנה מודלים הגידול vivo בשנים הבאות. יעילות הטיפול נקבע ביחס מינון התרופה, התוואי של הממשל שלה, העיתוי של הזרקת סמים ויישום של פולסים חשמליים, עוצמת השדה החשמלי, עם כיסוי שדה חשמלי גבוה מספיק (E), ואת הבחירה המתאימה של האלקטרודות ומיקום בכבוד אל הגידול 1,6,7,8. יתר על כן, המנגנונים את האפקטיביות של electrochemotherapy היו פירט, הוכחת כי, מלבד ההשפעה הישירה של electrochemotherapy לתאי הגידול, השפעה לשבש כלי הדם ואת התגובה החיסונית מעורבים 1,6,9. כל אלו נתונים שנאספו לאפשר תרגום של electrochemotherapy אל המרפאות. הניסויים הקליניים הראשונים הפגינו יעילות electrochemotherapy על הראש והצוואר מלנומה גושים הגידול 10. מאוחר יותר, יעילותו הודגם על סוגי גידולים אחרים, כגון קרצינומה של התאים הבזליים של העור, גרורות של מלנומה עורית, גידולים החלב, hypernephroma ו סרקומה של קפוסי. קיימים מספר דוחות הערכת באופן קולקטיבי את כל הנתונים הקליניים שפורסמו electrochemotherapy bleomycin עם ציספלטין ו 11-15. בסך הכל, שיעור התגובה של גידולים שטופלו היה כ 80% התגובות אובייקטיבי וכ התגובות להשלים 70% 15,16. היעילות ניתן עוד יותר על ידי טיפול חוזרות 17. כל אלה נתונים קליניים אפשרו electrochemotherapy להיות מאומץ בחלק ממדינות אירופה כמו הטיפול הסטנדרטי, מתוך כוונה פליאטיבי, על גידולים שונים. העתיד של טיפול זה היא להציג electrochemotherapy בטיפול של גידולים וגרורות פנימיים, בטיפול משולב, או עם טיפול גנטי או הקרנות. ניסיונות אלה כבר בעיצומן.

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<td>Bleomycin (Blenamax)</td>
<td>Reagent</td>
<td>Pharmachemie B.V.</td>
<td>&nbsp;</td>
<td>chemotherapeutic drug used in electrochemotherapy </td>
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<td>Cliniporator</td>
<td>Other</td>
<td>IGEA srl</td>
<td>&nbsp;</td>
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electrochemotherapy of tumours

Electrochemotherapy היא להשתמש בשילוב של תרופות כימותרפיות מסוימות פולסים חשמליים להחיל גולה הגידול המטופל. יישום מקומי של פולסים חשמליים אל הגידול עולה משלוח התרופה לתאים, במיוחד באתר של יישום פולס חשמלי. ספיגת התרופה על ידי מסירה של פולסים חשמליים הוא גדל רק אלה תרופות כימותרפיות אשר הובלה דרך הממברנה פלזמה נפגעת. בין תרופות רבות אשר נבדקו עד כה, מצאו bleomycin ו ציספלטין דרכם מניסוי קליני לשימוש קליני. נתונים קליניים שנאספו במסגרת מספר מחקרים קליניים מצביעים על כך כ -80% עורית המטופל ואת גושים תת עורי הגידול של גידולים ממאירים שונים בתגובה אובייקטיבי, מתוך אלה, כ -70% בתגובה להשלים לאחר יישום יחיד של electrochemotherapy. בדרך כלל רק טיפול אחד יש צורך, עם זאת, electrochemotherapy ניתן לחזור מספר פעמים כל כמה שבועות עם יעילות שווה כל הזמן. הטיפול מביא לחיסול יעיל של גושים מטופלים, עם אפקט קוסמטי טוב ללא הצטלקות הרקמה.

Watch this Scientific Journal Video about Electrochemotherapy of Tumours at JoVE.com

Electrochemotherapy of Tumours

Electrochemotherapy היא להשתמש בשילוב של תרופות כימותרפיות מסוימות, כגון ציספלטין ו bleomycin ו פולסים חשמליים להחיל גולה הגידול המטופל. אנו מציגים את פרוטוקול קליני של electrochemotherapy עבור treatmen של תת עורית ועל גרורות של מלנומה עורית.

Electrochemotherapy של גידולים

Electrochemotherapy is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of ...

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JoVE Journal

Biology

24.4K Views.  Institute of Oncology Ljubljana. Electrochemotherapy היא להשתמש בשילוב של תרופות כימותרפיות מסוימות, כגון ציספלטין ו bleomycin ו פולסים חשמליים להחיל גולה הגידול המטופל. אנו מציגים את פרוטוקול קליני של electrochemotherapy עבור treatmen של תת עורית ועל גרורות של מלנומה עורית.

Video: Electrochemotherapy of Tumours

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.

This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.

תיוג מדרדר לתאי הגנגליון של הרשתית על ידי יישום של זהב Fluoro על פני השטח של Colliculus מעולה

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for ...

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

בידוד של Protoplasts מרקמות של 14 יום בן שתילים של ארבידופסיס thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

יישום הופסק זרימה שיטות קינטיקס לחקור את מנגנון הפעולה של חלבון תיקון DNA

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Cryopreservation של עוברים Preimplantation של בקר, כבשים ועזים

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

הפעלה של אפופטוזיס על ידי Microinjection cytoplasmic של ציטוכרום ג</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

יישום MassSQUIRM עבור מדידות כמותיות של פעילות Demethylase ליזין

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

זיהוי לאחר translational השינויים של קומפלקסי חלבוני צמח

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

שיטה של ​​permeabilization של<em&gt; דרוזופילה</em&gt; עוברים למבחנים של מולקולה קטנה פעילות

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

שיטה להערכת ההשפעות של טווח של אורכי גל ועוצמות של אדום / טיפול באור האינפרה-אדום קרוב בסטרס חמצוני<em&gt; במבחנה</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.