Die Autoren danken für die finanzielle Unterstützung aus dem Staatshaushalt der slowenischen Research Agency und der Europäischen Kommission ESOPE Projekt (QLK-2002-02003), im Rahmen des 5. Rahmenprogramms finanziert. Die Autoren danken den Beitrag von prof. Marko Snoj, prof. Maja Cemazar, Matej Merkac und Rado Likon in Vorbereitung dieses Video-Artikel.

Die Behandlungsmethode für Elektrochemotherapie besteht aus lokalen oder systemischen Medikamenten Injektion durch Lieferung von elektrischen Impulsen angewendet, um den Tumor 1 gefolgt. Detaillierte Informationen über die Behandlung ist in Standard Operating Procedures (SOP) 2 veröffentlicht. Indikationen: <ol><li> Tumor-Typ: Basalzellkarzinom und Hautmetastasen von malignen Melanomen, Mammakarzinom, Hypernephrom, Kaposi-Sarkom und andere. </li><li> Tumor Ort: zugänglich kutanen und subkutanen Tumorknoten auf alle Teile des Körpers. </li><li> Tumorgröße kann von 3 mm bis 3-5 cm im Durchmesser sein. </li><li> Anzahl der Knoten, von 1 bis zu 100. </li></ol> Behandlungsablauf: <ol><li> Lokale Betäubung oder Vollnarkose, je nach der Anzahl der behandelten Knollen und die Lage. </li><li> Drug Wahl: Cisplatin oder Bleomycin. Cisplatin und Bleomycin kann intratumoral injiziert werden, Bleomycin auch intravenös in einer Dosis von 15000 IU / m 2. Injizierten Dosis für intratumorale Applikation von Bleomycin ist 250-1000 IU / cm 3 von Tumorgewebe und Cisplatin 0,5 -1 mg / cm 3 von Tumorgewebe </li><li> Zeitintervall zwischen den Drug Administration und Anwendung von elektrischen Impulsen, nach intratumorale Injektion des Medikaments sofort nach intravenöser Gabe nach 8 min. bis zu 28 min. </li><li> Elektrische Impulsgeber für Elektrochemotherapie sollte Rechteck elektrische Impulse mit einer Amplitude 1000 V oder mehr, Wiederholfrequenz von 1 Hz bis 5 kHz bieten. </li><li> Elektrische Pulsparameter: 8 Impulse von zB 1000 V bei 8 mm Abstand zwischen zwei Plattenelektroden, Frequenz 1 Hz oder 5 kHz, Pulsdauer 100 us. </li><li> Wenn der ganze Tumor kann nicht zwischen den Elektroden umfasst werden, sollte die Anwendung von elektrischen Impulsen wiederholt, dass viele Male, so dass die ganze Geschwulst Bereich, einschließlich Sicherheitsabstände zu decken. </li><li> Elektrodentyp: Plattenelektroden sind für kleine, oberflächliche Tumorknoten verwendet werden Nadelelektroden für die Behandlung von tiefer gesetzte Knötchen unter der Haut. </li></ol> Weitere Merkmale der Behandlung: <ol><li> In der Regel einzelne Behandlung ist für Tumorknoten Ausrottung, aber Elektrochemotherapie kann in 4-Wochen-Intervallen auf die restlichen Tumorgewebe oder andere Tumorknoten wiederholt werden. </li></ol> Aufgrund fehlender systemischer Toxizität - weil sehr niedrigen Dosen von Bleomycin oder Cisplatin sind notwendig - Patienten können auf einer ambulant behandelt werden, und kann das Krankenhaus bald verlassen nach der Behandlung.

<ol>
	<li>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=Bases+and+rationale+of+the+electrochemotherapy.">Bases and rationale of the electrochemotherapy.</a> EJC Suppl. 4, 38-44 (2006).</li><li>Mir, L. M., Gehl, J., Sersa, G., Collins, C. G., Garbay, J. R., Billard, V., Geertsen, P., Rudolf, Z., O'Sullivan, G. C., Marty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+operating+procedures+of+the+electrochemotherapy:+instructions+for+the+use+of+bleomycin+or+cisplatin+administered+either+systemically+or+locally+and+electric+pulses+delivered+by+the+CliniporatorTM+by+means+of+invasive+or+non-invasive+electrodes.">Standard operating procedures of the electrochemotherapy: instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes.</a> EJC Suppl. 4, 14-25 (2006).</li><li>Okino, M., Mohri, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high-voltage+electrical+impulse+and+an+anticancer+drug+on+in+vivo+growing+tumors.">Effects of high-voltage electrical impulse and an anticancer drug on in vivo growing tumors.</a> Jpn. J. Cancer Res. 78, 1319-1321 (1987).</li><li>Mir, L. M., Banoun, H., 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=Introduction+of+definite+amounts+of+nonpermeant+molecules+into+living+cells+after+electropermeabilization:+direct+access+to+the+cytosol.">Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol.</a> Exp. Cell Res. 175, 15-25 (1988).</li><li>Sersa, G., Cemazar, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effectiveness+of+electrochemotherapy+with+cis-diamminedichloroplatinum(II)+in+mice.">Antitumor effectiveness of electrochemotherapy with cis-diamminedichloroplatinum(II) in mice.</a> Cancer Res. 55, 3450-3455 (1995).</li><li>Sersa, G., Cemazar, M., Miklavcic, D., Rudolf, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemotherapy+of+tumours.">Electrochemotherapy of tumours.</a> Radiol. Oncol. 40, 163-174 (2006).</li><li>Miklavcic, D., Beravs, K., Semrov, D., Cemazar, M., Demsar, F., Sersa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+electric+field+distribution+for+effective+in+vivo+electroporation+of+tissues.">The importance of electric field distribution for effective in vivo electroporation of tissues.</a> Biophys. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+metastatic+melanoma+using+electroporation+therapy+with+bleomycin+(electrochemotherapy).">Treatment of metastatic melanoma using electroporation therapy with bleomycin (electrochemotherapy).</a> Melanoma Res. 15, 45-51 (2005).</li><li>Sersa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state-of-the-art+of+electrochemotherapy+before+the+ESOPE+study:+advantages+and+clinical+uses.">The state-of-the-art of electrochemotherapy before the ESOPE study: advantages and clinical uses.</a> EJC Suppl. 4, 52-59 (2006).</li><li>Sersa, G., Miklavcic, D., Cemazar, M., Rudolf, Z., Pucihar, G., Snoj, 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+in+treatment+of+tumours.">Electrochemotherapy in treatment of tumours.</a> EJSO. 34, 232-240 (2008).</li><li>Marty, M., Sersa, G., Garbay, J. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemotherapy+with+intravenous+bleomycin+in+the+local+treatment+of+skin+melanoma+metastases.">Electrochemotherapy with intravenous bleomycin in the local treatment of skin melanoma metastases.</a> Ann. Surg. Oncol. 15, 2215-2222 (2008).</li></ol>

Erhöhung der Zytotoxizität von Bleomycin durch Exposition von Zellen gegenüber electroporative elektrische Impulse wurde zuerst von Okino M und Mir LM 3,4 beschrieben. Danach Elektrochemotherapie konnte gezeigt werden, um wirksam zu sein auch für Cisplatin 5. Umfangreiche präklinische Daten wurden auf in vitro und in vivo Tumormodellen in den folgenden Jahren gesammelt. Wirksamkeit der Behandlung wurde im Zusammenhang mit Drogenkriminalität Dosierung, Art der Verwaltung, Timing der Injektion und Anwendung von elektrischen Impulsen, die Intensität des elektrischen Feldes, Abdeckung mit einer ausreichend hohen elektrischen Feld (E) und geeignete Wahl der Elektroden und Positionierung gegenüber bestimmt um den Tumor 1,6,7,8. Darüber hinaus wurden Mechanismen der Wirksamkeit der Elektrochemotherapie erarbeitet, die zeigen, dass, neben den direkten Effekt von Elektrochemotherapie an Tumorzellen, gefäßzerstörenden Wirkung und Immunantwort sind 1,6,9 beteiligt. Alle diese gesammelten Daten aktiviert Übersetzung Elektrochemotherapie in den Kliniken. Die ersten klinischen Studien nachgewiesenen Wirksamkeit von Elektrochemotherapie auf Kopf und Hals und Melanom Tumorknoten 10. Später wurde seine Wirksamkeit bei anderen Tumorarten, wie Basalzellkarzinom der Haut, kutane Metastasen von Melanom, Mamma-Tumoren, Hypernephrom und Kaposi-Sarkom unter Beweis gestellt. Es gibt mehrere Berichte zur Bewertung kollektiv alle klinischen Daten über Elektrochemotherapie mit Bleomycin und Cisplatin 11-15 veröffentlicht. Insgesamt war die Rücklaufquote der behandelten Tumoren etwa 80% objektives Ansprechen und ca. 70% komplette Remissionen 15,16. Die Wirksamkeit kann sogar noch höher durch wiederholte Behandlung 17. Alle diese klinischen Daten haben Elektrochemotherapie aktivieren, damit Sie in einigen europäischen Ländern als Standard-Behandlung angenommen werden, mit palliativer Intention, auf verschiedenen Tumoren. Die Zukunft dieser Behandlung ist es, Elektrochemotherapie bei der Behandlung von inneren Tumoren und Metastasen, und in der kombinierten Behandlung einzuführen, entweder mit Gentherapie oder Strahlentherapie. Diese Versuche sind bereits im Gange.

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<td>Bleomycin (Blenamax)</td>
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<td>chemotherapeutic drug used in electrochemotherapy </td>
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<td>Cliniporator</td>
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electrochemotherapy of tumours

Elektrochemotherapie ist eine kombinierte Verwendung bestimmter Chemotherapeutika und elektrischen Impulsen, die behandelten Tumors Knötchen. Die lokale Anwendung von elektrischen Impulsen, um den Tumor erhöht Drug Delivery in Zellen, insbesondere an der Stelle des elektrischen Impulses Anwendung. Drug-Aufnahme durch Lieferung der elektrischen Impulse ist nur für die Chemotherapeutika, deren Transport durch die Plasmamembran behindert wird erhöht. Unter den vielen Medikamenten, die getestet wurden bisher gefunden Bleomycin und Cisplatin ihrem Weg von vorklinischen Tests für die klinische Anwendung. Klinische Daten in einer Reihe von klinischen Studien gesammelten Daten zeigen, dass ca. 80% der behandelten kutanen und subkutanen Tumorknoten verschiedener Malignome in eine objektive Ansprechrate sind, von diesen, etwa 70% in komplette Remission nach einer einzigen Anwendung Elektrochemotherapie. Üblicherweise wird nur eine Behandlung erforderlich ist, kann jedoch Elektrochemotherapie mehrmals wiederholt werden, alle paar Wochen mit gleicher Effektivität jeder Zeit. Die Behandlung führt zu einer effektiven Bekämpfung der behandelten Knollen, mit einem guten kosmetischen Effekt, ohne Gewebe Narben.

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

Electrochemotherapy of Tumours

Elektrochemotherapie ist eine kombinierte Verwendung von bestimmten Chemotherapeutika wie Cisplatin und Bleomycin und elektrischen Impulsen, die behandelten Tumors Knötchen. Wir präsentieren die klinischen Protokoll Elektrochemotherapie für treatmen der subkutanen und kutanen Metastasen von Melanomen.

Elektrochemotherapie von Tumoren

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

Research

JoVE Journal

Biology

24.4K Aufrufe.  Institute of Oncology Ljubljana. Elektrochemotherapie ist eine kombinierte Verwendung von bestimmten Chemotherapeutika wie Cisplatin und Bleomycin und elektrischen Impulsen, die behandelten Tumors Knötchen. Wir präsentieren die klinischen Protokoll Elektrochemotherapie für treatmen der subkutanen und kutanen Metastasen von Melanomen.

Serie: 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.

Retrograde Markierung von retinalen Ganglienzellen durch Anwendung von Fluoro-Gold auf der Oberfläche des Colliculus superior

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

Forschung

Biologie

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.

Isolation von Protoplasten aus Geweben von 14-Tage alte Keimlinge von Arabidopsis 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.

Die Anwendung der Stopped-Flow-Kinetics Methoden der Wirkmechanismus eines DNA-Repair Protein Untersuchen

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.

Kryokonservierung von Präimplantationsembryonen von Rindern, Schafen und Ziegen

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.

Die Aktivierung der Apoptose durch cytoplasmatische Mikroinjektion von Cytochrom C</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.

Die Anwendung des MassSQUIRM für quantitative Messungen von Lysin-Demethylase

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.

Bezeichnung des Post-translationale Modifikationen von Pflanzenproteinkomplexe

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.

Ein Verfahren zur Permeabilisierung der<em&gt; Drosophila</em&gt; Embryonen für Tests von Small Molecule Aktivität

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.

Verfahren zur Beurteilung der Auswirkungen einer Reihe von Wellenlängen und Intensitäten von Rot / Nah-Infrarot-Licht-Therapie auf oxidativen Stress<em&gt; In-vitro-</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.