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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J. 74, 2152-2158 (1998).</li><li>Miklavcic, D., Corovic, S., Pucihar, G., Pavselj, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+tumour+coverage+by+sufficiently+high+local+electric+field+for+effective+electrochemotherapy.">Importance of tumour coverage by sufficiently high local electric field for effective electrochemotherapy.</a> EJC Suppl. 4, 45-51 (2006).</li><li>Sersa, G., Jarm, T., Kotnik, T., Coer, A., Podkrajsek, M., Sentjurc, M., Miklavcic, D., Kadivec, M., Kranjc, S., Secerov, A., Cemazar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+disrupting+action+of+electroporation+and+electrochemotherapy+with+bleomycin+in+murine+sarcoma.">Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma.</a> Brit. J. 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. M., DeConti, R., Stabuc, B., Cemazar, M., Coninx, P., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+cutaneous+and+subcutaneous+malignant+tumours+by+electrochemotherapy.">Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy.</a> Brit. J. Cancer. 77, 2336-2342 (1998).</li><li>Gothelf, A., Mir, L. M., Gehl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemotherapy:+results+of+cancer+treatment+using+enhanced+delivery+of+bleomycin+by+electroporation.">Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation.</a> Cancer Treat. Rev. 29, 371-387 (2003).</li><li>Byrne, C. M., Thompson, J. F., Johnston, H., Hersey, P., Quinn, M. J., Hughes, M., McCarthy, W. 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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>
</tr>
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<td>Cliniporator</td>
<td>Other</td>
<td>IGEA srl</td>
<td>&nbsp;</td>
<td>Generator of electric pulses</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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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

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 ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

Dissection ברוטו של קיבה של לובסטר, Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

ניתוח ופיתוח של הפנוטיפ מורפולוגי כפונקציה של ריכוז חלבון שמרים הנצה

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

קביעת מתילציה DNA של גנים מוטבעים ארבידופסיס האנדוספרם

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

השתלה של תאים ישירות לתוך הכליה של דג הזברה למבוגרים

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

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 ...

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 ...