The authors gratefully acknowledge financial support from the state budget of the Slovenian Research Agency and the European Commission's ESOPE project (QLK-2002-02003), funded under the 5th Framework Programme. The authors acknowledge the contribution of prof. Marko Snoj, prof. Maja Cemazar, Matej Merkac and Rado Likon in preparation of this Video-article.

The treatment procedure for electrochemotherapy consists of local or systemic drug injection followed by delivery of electric pulses applied to the tumour 1. Detailed information about the treatment is published in Standard Operating Procedures (SOP) 2.
Treatment indications:
<ol>
<li>Tumour type: basal cell carcinoma, and cutaneous metastases of malignant melanoma, breast carcinoma, hypernephroma, Kaposi's sarcoma and others.</li>
<li>Tumour location: accessible cutaneous and subcutaneous tumour nodules on all parts of the body.</li>
<li>Tumour size can be from 3 mm to 3-5 cm in diameter.</li>
<li>Number of nodules; from 1 to up to 100.</li>
</ol>
Treatment procedure:
<ol>
<li>Local or general anaesthesia, depending on the number of the treated nodules and the location.</li>
<li>Drug choice: cisplatin or bleomycin. Cisplatin and bleomycin can be injected intratumorally, bleomycin also intravenously in a dose of 15000 IU/m2. Injected dose for intratumoural application of bleomycin is 250-1000 IU/cm3 of tumour tissue and for cisplatin 0.5 -1 mg/cm3 of tumour tissue </li>
<li>Time interval between the drug administration and application of electric pulses; after intratumoral injection of the drug immediately, after intravenous administration after 8 min. up to 28 min.</li>
<li>Electric pulse generator for electrochemotherapy should provide square wave electric pulses of amplitude 1000 V or more, repetition frequency from 1 Hz to 5 kHz.</li>
<li>Electric pulse parameters: 8 pulses of e.g. 1000 V at 8 mm distance between two plate electrodes, frequency 1 Hz or 5 kHz, duration of the pulse 100 &micro;s.</li>
<li>If the whole tumour can not be encompassed between the electrodes, application of electric pulses should be repeated that many times so as to cover the whole tumour area, including safety margins.</li>
<li>Electrode type: plate electrodes are used for small, superficial tumour nodules, needle electrodes are for treatment of deeper seeded nodules under the skin.</li>
</ol>
Other features of the treatment:
<ol>
<li>Usually single treatment is effective for tumour nodule eradication; however electrochemotherapy can be repeated in 4 weeks intervals on the remaining tumour tissue or other tumour nodules.</li>
</ol>
Due to lack of systemic toxicity - because very low doses of bleomycin or cisplatin are needed - patients can be treated on an out-patient basis, and can leave the hospital soon after the treatment.

<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>

Increase in cytotoxicity of bleomycin by exposure of cells to electroporative electric pulses was first described by Okino M and Mir LM 3,4. Thereafter electrochemotherapy was demonstrated to be effective also for cisplatin 5. Extensive preclinical data were collected on in vitro and in vivo tumour models in the following years. Treatment effectiveness was determined in relation to drug dosage, route of its administration, timing of drug injection and application of electric pulses, intensity of the electric field, coverage with sufficiently high electric field (E), and appropriate selection of electrodes and positioning with respect to the tumour 1,6,7,8. Furthermore, mechanisms underlying the effectiveness of electrochemotherapy were elaborated, demonstrating that, besides direct effect of electrochemotherapy to tumour cells, vascular disrupting effect and immune response are involved 1,6,9. All of these collected data enabled translation of electrochemotherapy into the clinics.
The first clinical trials demonstrated effectiveness of electrochemotherapy on head and neck and melanoma tumour nodules 10. Later on, its effectiveness was demonstrated on other tumour types, such as basal cell carcinoma of the skin, cutaneous metastases of melanoma, mammary tumours, hypernephroma and Kaposi's sarcoma. There are several reports evaluating collectively all clinical data published on electrochemotherapy with bleomycin and cisplatin 11-15. Overall, the response rate of the treated tumours was approximately 80% objective responses and approximately 70% complete responses 15,16. The effectiveness can be even higher by repetitive treatment 17.
All of these clinical data have enabled electrochemotherapy to be adopted in some European countries as standard treatment, with palliative intent, on various tumours. The future of this treatment is to introduce electrochemotherapy in treatment of internal tumours and metastases, and in combined treatment, either with gene therapy or radiation therapy. These attempts are already under way.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bleomycin (Blenamax)</td>
<td>Reagent</td>
<td>Pharmachemie B.V.</td>
<td>&nbsp;</td>
<td>chemotherapeutic drug used in electrochemotherapy </td>
</tr>
<tr>
<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 is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of electric pulses to the tumour increases drug delivery into cells, specifically at the site of electric pulse application. Drug uptake by delivery of electric pulses is increased for only those chemotherapeutic drugs whose transport through the plasma membrane is impeded. Among many drugs that have been tested so far, bleomycin and cisplatin found their way from preclinical testing to clinical use. Clinical data collected within a number of clinical studies indicate that approximately 80% of the treated cutaneous and subcutaneous tumour nodules of different malignancies are in an objective response, from these, approximately 70% in complete response after a single application of electrochemotherapy. Usually only one treatment is needed, however, electrochemotherapy can be repeated several times every few weeks with equal effectiveness each time. The treatment results in an effective eradication of the treated nodules, with a good cosmetic effect without tissue scarring.

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

Electrochemotherapy of Tumours

Electrochemotherapy is a combined use of certain chemotherapeutic drugs such as cisplatin and bleomycin and electric pulses applied to the treated tumour nodule. We present the clinical protocol of electrochemotherapy for treatmen of subcutaneous and cutaneous metastases of melanoma.

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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24.4K Views.  Institute of Oncology Ljubljana. Electrochemotherapy is a combined use of certain chemotherapeutic drugs such as cisplatin and bleomycin and electric pulses applied to the treated tumour nodule. We present the clinical protocol of electrochemotherapy for treatmen of subcutaneous and cutaneous metastases of melanoma. 

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.

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.

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

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.

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.

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.

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.

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.

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.

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

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...