Ricin-cytotoxin conjugate administration reveals a physiologically relevant role for oxytocin in the control of gonadotropin secretion.
Ricin-cytotoxin conjugate administration reveals a physiologically relevant role for oxytocin in the control of gonadotropin secretion.
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蓖麻毒素-细胞毒素缀合物的施用揭示了催产素在控制促性腺激素分泌中的生理相关作用。
DOI：
10.1111/j.1749-6632.1992.tb34371.x
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发表时间：
1992
影响因子：
5.2
通讯作者：
Fulton,RJ
中科院分区：
文献类型：
--
作者：
Samson,WK;Alexander,BD;Skala,KD;Huang,FL;Fulton,RJ
方法用异双功能交联剂N-琥珀酰亚胺3-(2-吡啶二硫)丙酸酯(SPDP，皮尔斯化学公司，罗克福德，伊利诺伊州，伊利诺伊州)，通过二硫键将多肽(半岛实验室，贝尔蒙特，加利福尼亚州贝尔蒙特)通过二硫键连接到脱糖富含A链(内陆实验室，奥斯汀，德克萨斯州)。I6富A链(在PBS中为5 mg/ml)在室温下被四倍摩尔过量的SPDP(在二甲基甲酰胺中为10 mg/ml)取代30min，然后用5.0 nM二硫苏糖醇在室温下还原30min，从而平均引入两个额外的自由巯基。然后在Sephadex(3-25(Pharmacia/LKB，Piscataway，NJ))柱上脱盐，在PBS中平衡。将多肽溶解在磷酸盐缓冲盐水(PBS)中，并在室温下用四倍摩尔的SPDP取代30min，然后在Biogel P2(BioRad，里士满，加利福尼亚州)柱上分离，以去除多余的交联剂。然后将多肽和A链以每摩尔A链四个摩尔肽的比例混合，并在4℃下孵育16小时。反应在G-25柱上脱盐，在PBS中平衡，以去除未结合的多肽。结合物无菌过滤并储存在-20℃的等量中(类固醇来自Sigma Chemical Co.[苯甲酸雌二醇酯，圣路易斯，密苏里州]和Schein PharmPharmticals[黄体酮，纽约州华盛顿港])。成年雌性大鼠(体重200-225g，Spraogue-Dawley，Sasco，Omaha，NE)被单独饲养在受控条件下(21-23“℃，光照0500-1900小时，随意自来水和实验室食物)。所有程序都得到了密苏里大学-哥伦比亚大学动物保护和使用委员会的批准。对于活体方案，如前所述，在第三脑室立体定向植入一个长期留置的不锈钢套管(23号)6，要么是通过监测阴道涂片建立正常发情周期的完整大鼠，要么是在双侧卵巢切除一周后在其他大鼠身上植入。在循环动物中，在发情前期的早晨，在轻度乙醚麻醉下注射蓖麻毒素A链(1um1生理盐水，非特异性毒素对照)、富含LHRH的A链结合物(1 mg/m l生理盐水，多肽对照)或OT-蓖麻毒素A链结合物(1 mg/m l生理盐水)。在注射后至少45天内每天进行阴道涂片，以确定下一次连续发情和发情涂片出现的时间间隔和周期模式。在一组去卵巢(OVX)的大鼠中，在性腺切除后两周进行了类似的注射。7天后，在50p1玉米油中注入5pg苯甲酸雌二醇钠，第二天，植入留置颈静脉插管，以便于从清醒的、自由的动物身上采集血液。24小时后(100小时)，所有大鼠在50pl芝麻油中注射黄体酮SC 2 mg，并将一根延长管(充满肝素化的生理盐水)连接到颈静脉插管。在不干扰动物的情况下，每小时从1200至1800小时通过插管抽取血样(0.5毫升)。取血量用0.9%氯化钠代替。OVX组大鼠在脑室插管后1周注射未结合的蓖麻毒素A链(3p1生理盐水载体)、富OT的A链(3p1载体3pg)或生理盐水载体(3pl)，7天后再次注射。与第一组相同(见上文)进行类固醇预注射，并在1200h和1400h至1900h之间每小时采集一次血液样本。…
METHODSPeptides (Peninsula Laboratories, Belmont, CA) were conjugated to deglycosylated rich A chain (Inland Laboratories, Austin, TX) via disulfide linkage using the heterobifunctional cross-linker, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pierce Chemical Co., Rockford, IL) by a modification of the method used for preparing antibody-ricin A chain conjugates. I6 Rich A chain (5 mg/ml in PBS) was thiolated to introduce an average of two additional free sulfhydryl groups by substitution with a fourfold molar excess of SPDP (10 mg/ml in dimethyl formamide) for 30 min at room temperature, followed by reduction with 5.0 nM dithiothreitol for 30 min at room temperature. The reaction was then desalted on a column of Sephadex (3-25 (Pharmacia/LKB, Piscataway, NJ) equilibrated in PBS. Peptides were dissolved in phosphate-buffered saline (PBS) and substituted with a fourfold molar excess of SPDP for 30 min at room temperature, followed by separation on a column of Biogel P2 (BioRad, Richmond, CA) to remove excess cross-linker. The peptide and A chain were then mixed at a ratio of four moles peptide per mole A chain and incubated 16 hours at 4" C. The reaction was desalted on a G-25 column, equilibrated in PBS, to remove unconjugated peptide. Conjugates were sterile filtered and stored in aliquots at-20" C.(Steroids were obtained from Sigma Chemical Co.[estradiol benzoate, St. Louis, MO] and Schein Pharmaceuticals [progesterone, Port Washington, NY].) Adult, female rats (200-225 g, Sprague-Dawley, Sasco, Omaha, NE) were housed individually under controlled conditions (21-23" C, lights on 0500-1900 hr, ad libitum tap water and lab chow). All procedures were approved by the University of Missouri-Columbia Animal Care and Use Committee. For in vivo protocols a chronic, indwelling, stainless steel cannula (23 gauge) was stereotaxically implanted in the third cerebroventricle as previously described6 either in intact rats after establishment of normal estrous cyclicity by monitoring vaginal smears or in other rats one week after bilateral ovariectomy. In cycling animals, 3-pl injections of ricin A chain (1 mum 1 saline, as nonspecific toxin control), LHRH-rich A chain conjugate (1 mg/ml saline, peptide control), or OT-ricin A chain conjugate (1 mg/ml saline) were conducted on the morning of proestrus under light ether anesthesia. Vaginal smears were taken daily for a minimum of 45 days after injections to establish the time interval to the appearance of the next consecutive proestrous and estrous smears and the pattern of cyclicity. In one group of ovariectomized (OVX) rats, similar injections were conducted two weeks after gonadectomy. Seven days later, 5 pg estradiol benzoate in 50 p1 corn oil was injected SC On the following day an indwelling jugular cannula was implanted to facilitate blood sampling from conscious, unrestrained animals. Twenty-four hours later (loo0 hr) all rats were injected with 2 mg progesterone SC in 50 pl sesame oil and an extension tubing (filled with heparinized saline) was attached to the jugular cannula. Blood samples (0.5 ml) were withdrawn via the cannula, without disturbing the animals, at hourly intervals from 1200 to 1800 hr. The volume of blood removed was replaced with 0.9% NaCl. In a second group of OVX rats, unconjugated ricin A chain (3 pg in 3 p1 saline vehicle), OT-rich A chain (3 pg in 3 p1 vehicle), or saline vehicle alone (3 pl) were injected one week after implantation of the cerebroventricular cannula and again seven days later. Steroid priming was conducted as in the first group (vida supra), and blood samples collected at 1200 hr and at hourly intervals between 1400 and 1900 hr …