作者非常感谢斯洛文尼亚研究机构的国家预算和欧洲委员会的ESOPE项目（QLK - 2002 - 02003），根据第五框架计划资助的的财政支持。作者承认教授的贡献。马尔科Snoj，教授。玛雅Cemazar，Matej Merkac和雷达Likon此视频文章做准备。

电化学治疗过程由局部或全身药物注射交付应用到肿瘤的1电脉冲。关于治疗的详细信息公布在标准操作程序（SOP）2 。 治疗适应症： <ol><li>肿瘤类型：基底细胞癌和皮肤恶性黑色素瘤，乳腺癌，hypernephroma，卡波西氏肉瘤和其他转移。 </li><li>肿瘤的位置：访问的皮肤和身体的各个部位的皮下肿瘤结节。 </li><li>肿瘤的大小，可从3毫米，直径3-5厘米。 </li><li>结节数从1到100。 </li></ol>治疗过程： <ol><li>局部或全身麻醉，这取决于治疗结节的数目和位置。 </li><li>药物的选择：顺铂或博莱霉素。瘤内注入顺铂和博莱霉素，博莱霉素也静脉注射剂量在15000 IU / M 2。 intratumoural应用博莱霉素注射剂量为250-1000 IU /厘米3的肿瘤组织，顺铂0.5 -1毫克/厘米的肿瘤组织 3 </li><li>药品监督管理和应用的电脉冲之间的时间间隔;瘤内注射药物后，静脉给药后8分钟后立即。长达28分钟。 </li><li>电脉冲电化学发电机应提供从1 Hz到5 kHz的方波振幅1000伏以上，重复频率的电脉冲。 </li><li>电脉冲参数：如1000伏的脉冲，在两个电极板之间，频率为1 Hz或5千赫，脉冲持续时间100微秒的8毫米的距离。 </li><li>如果整个肿瘤不能包含两个电极之间的电脉冲应用程序应反复多次，以覆盖整个肿瘤区，包括安全边际。 </li><li>电极类型：小，浅表肿瘤结节电极板，电极针治疗更深种子结节在皮肤下。 </li></ol>治疗的其他特点： <ol><li>通常单一的治疗方法是有效地消除肿瘤结节;然而，电化学可以在剩下的肿瘤组织或其他肿瘤结节4周的时间间隔反复。 </li></ol>由于缺乏全身毒性 - 博莱霉素或顺铂剂量很低，因为需要 - 患者可以治疗门诊的基础上，可以离开医院治疗后很快。

<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>

最初是由冲之M和和平号空间站的LM 3,4 electroporative电脉冲的细胞暴露在博莱霉素的细胞毒性增加。此后电化学被证明是有效的顺铂5。在接下来的几年广泛的临床前数据收集在体外和体内肿瘤模型。治疗效果取决于药物剂量，其管理的途径，药物注射和电脉冲的应用程序的时间，电场强度，具有足够高的电场（E）的覆盖面，并选择适当的电极和定位方面肿瘤1,6,7,8。此外，内在机制的电化学的有效性进行了详细阐述，这表明，除了直接影响到肿瘤细胞，血管破坏作用和免疫反应的电化学涉及 1,6,9 。所有这些收集的数据使电化学翻译成诊所。 第一临床试验表明，头部和颈部和黑色素瘤结节 10电化学成效。后来，其效益是其他类型的肿瘤，如皮肤基底细胞癌，黑色素瘤皮肤转移，乳腺肿瘤，hypernephroma和卡波济氏肉瘤，证明。有评估集体所有临床数据上的电化学与博莱霉素，顺铂11-15出版的几份报告。总体而言，治疗肿瘤的反应率约80％的客观反应和大约70％ 的 15,16完整的答复。有效性，可重复治疗17甚至更高。 所有这些临床数据已启用电化学在欧洲一些国家采用作为标准治疗，治标不治本的意图，对各种肿瘤。这种治疗方法的未来，是引入内部的肿瘤和转移，处理和电化学综合治疗，基因治疗或放射治疗。这些尝试已经在进行之中。

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		<th>Material Name</th>
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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>
<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

电化学是一个结合使用某些化疗药物应用于治疗肿瘤结节的电脉冲。当地的电脉冲肿瘤中的应用，增加进入细胞的药物输送，特别是在电脉冲应用的网站。只有那些其运输通过质膜是阻碍化疗药物增加药物的吸收是电脉冲交付。其中许多药物，至今已经过测试，博莱霉素，顺铂发现从临床前试验，临床使用的方式。在临床研究中收集的临床数据表明，约80％的治疗不同恶性肿瘤的皮肤和皮下肿瘤结节在客观反应，从这些，大约有70％完全缓解后的电化学单一的应用程序。通常只有一个治疗是必要的，然而，电化学，可反复几次具有同等效力每次每隔几个星期。在一个有效的根除治疗结节的治疗效果，具有良好的无组织疤痕的美容效果。

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

Electrochemotherapy of Tumours

电化学是如某些化疗药物顺铂和博莱霉素应用于治疗肿瘤结节的电脉冲的联合使用。我们目前对皮下黑色素瘤皮肤转移treatmen电化学临床协议。

电化学的肿瘤

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 次观看.  Institute of Oncology Ljubljana. 电化学是如某些化疗药物顺铂和博莱霉素应用于治疗肿瘤结节的电脉冲的联合使用。我们目前对皮下黑色素瘤皮肤转移treatmen电化学临床协议。

视频: 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.

上丘表面上应用氟金逆行标记视网膜神经节细胞

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

科研

生物学

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.

从14日龄的幼苗组织原生质体的分离拟南芥</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.

停流的动力学方法的应用研究的一种DNA修复蛋白的行动机制

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.

植入前胚胎，牛，羊，山羊的冷冻保存

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.

激活细胞凋亡的细胞色素的细胞质显微注射 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.

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

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.

植物蛋白复合体的翻译后修饰的鉴定

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.

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

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.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</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.

Methods Article

电化学的肿瘤