Enzymes of uridine 5'-monophosphate biosynthesis in Schistosoma mansoni

In Schistosoma mansoni, the major product of in vitro orotate metabolism was orotidine 5'-monophosphate (OMP), whereas in mouse liver it was UMP. In contrast to mammalian cells, OMP appeared not to be 'channeled' from orotate phosphoribosyltransferase to OMP decarboxylase in S. mansoni, resulting in substantial degradation of OMP to orotidine. Significant differences were observed in the inhibitor specificity of phosphoribosyltransferase between S. mansoni and mouse liver, indicating that this enzyme may be a potential chemotherapeutic target in S. mansoni. Two distinct phosphoribosyltransferases were found in S. mansoni. One enzyme, having the higher molecular weight, utilized orotate, 5-fluorouracil and uracil as substrates, while the other only orotate. Both enzymes were inhibited by 5-azaorotic acid (oxonic acid) but only the 'orotate-specific' enzyme was inhibited by 4,6-dihydroxypyrimidine. OMP decarboxylase activity co-eluted with both phosphoribosyltransferases from Sephadex G-100 gel chromatography. We suggest that phosphoribosyltransferase in S. mansoni plays a role in both de novo UMP biosynthesis as well as in the salvage of uracil and uridine.     

联合胞嘧啶脱胺酶与尿嘧啶磷酸核糖转移酶基因治疗小鼠胃癌

目的探讨联合应用尿嘧啶磷酸核糖转移酶(UPRT)基因对胞嘧啶脱胺酶(CD)/氟胞嘧啶(5-FC)前药转换酶系统抗瘤作用的增强效应。方法将携带UPRT基因和CD基因的逆转录病毒载体通过PA317细胞包装成完整的病毒颗粒并浓缩和纯化。建立615小鼠胃癌皮下移植瘤模型,瘤内反复多次注射携带UPRT和CD基因的逆转录病毒,同时腹腔内给予5-FC,观察抑瘤效果。结果逆转录病毒介导CD基因瘤内原位转导,同时联合应用5-FC进行治疗,结果发现,在肿瘤接种后第12天,对照组肿瘤体积为(2.367±0.371)cm3,而CD/5-FC治疗组肿瘤体积大小为(0.704±0.358)cm3,肿瘤生长明显受抑(P<0.0005)。联合转导UPRT基因后,于肿瘤接种后第16天,肿瘤体积为(0.731±0.255)cm3,而单独应用CD基因治疗组肿瘤体积为(1.455±0.370)cm3,从而证实联合转导UPRT基因可进一步增强CD/5-FC自杀基因系统的抑瘤作用(P<0.001)。结论联合转导UPRT基因,可显著增强CD/5-FC自杀基因系统的抗瘤效应。     

Chemoradiotherapy and adjuvant chemotherapy for rectal cancer

Local recurrence is an important factor in determining the outcome of patients after surgery for rectal cancer, and various attempts have been made to reduce the local recurrence rate. Randomized controlled trials have shown that radiotherapy combined with total mesorectal excision can reduce the local recurrence rate in rectal cancer patients who undergo curative surgery. Chemoradiotherapy is more effective in achieving local control than radiotherapy alone, and preoperative chemoradiotherapy is superior to postoperative chemoradiotherapy in terms of adverse events. Recent advances have led to the identification of potential therapeutic targets such as epidermal growth factor receptor, vascular endothelial growth factor, and endothelial receptors. These new agents have been used in combination with conventional chemoradiotherapy, and higher pathological complete response rates have been reported for such combinations in comparison with conventional regimens. With regard to lateral node dissection, a recent study showed that postoperative chemoradiotherapy was more effective in reducing the local recurrence rate than lateral node dissection. As for adjuvant chemotherapy, one randomized controlled trial showed that patients who received uracil and tegafur as adjuvant therapy had significantly prolonged relapse-free survival times and overall survival times. As well, one metaanalysis has shown the efficacy of oral uracil-tegafur as adjuvant chemotherapy for rectal cancer.     

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Purpose: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. Methods: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. Results: Following a 24-h treatment with 100 μM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80  C and 80  K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. Conclusions: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines. Received: 19 August / Accepted: 16 December 1997     

瘤内原位转导UPRT基因治疗小鼠胃癌的实验研究

背景与目的:5-FU是临床上重要的化疗药物,但总体疗效有限,如何提高其抗癌疗效一直是临床上亟待解决的难题。本研究采用基因治疗的原理与方法,探讨UPRT(uracilphosphoribosyltransferase)基因对5-FU杀伤效应的增敏作用。方法:采用PCR技术从大肠杆菌K12基因组中扩增UPRT基因,并构建pLXSN逆转录病毒表达载体,进一步转染小鼠胃癌细胞株MFC。采用MTT法检测转导UPRT基因前后MFC对5-FU敏感性的变化。建立615小鼠胃癌皮下移植瘤模型,瘤内反复多次注射携带UPRT基因的浓缩、纯化的逆转录病毒,同时腹腔内给予5-FU,观察抑瘤效果。结果:体外MTT法检测结果显示,转导UPRT基因后可使MFC对5-FU的敏感性提高约17.26倍。以逆转录病毒介导UPRT基因瘤内原位转导,同时联合应用5-FU进行治疗,结果肿瘤生长明显受抑(P<0.005),抑瘤率为87.18%。结论:UPRT基因修饰小鼠胃癌细胞株MFC,能明显提高MFC对5-FU杀伤的敏感性,以逆转录病毒载体介导UPRT基因肿瘤原位基因转导,同时联合应用5-FU,能获得良好的抑瘤效应。     

HPLC法测定血浆中尿嘧啶和二氢尿嘧啶的含量

目的：建立人血浆中内源性尿嘧啶（U）和二氢尿嘧啶（UH2）的HPLC测定方法，为通过UH2／U比值评价体内二氢嘧啶脱氢酶活性提供方法学基础。方法：采用Shim-packCLC—ODS柱（6mm×150mm，5μm），以0．05mol·L^-1磷酸盐缓冲液（pH值为4．0）为流动相，测定U的检测波长为260nm，测定UH2的检测波长205nm。以含3％牛血清白蛋白（BSA）的水溶液配制标准样品和质控样品。以溴尿嘧啶为内标，采用叔丁基甲醚／正丙醇（75：25）为提取溶剂进行两次提取，氮气吹干、复溶后进样分析。结果：U在5—200μg·L^-1的范围内线性良好（r=0．9995，n=6），测定的批内变异RSD在1．73％-3．45％之间，批间变异RSD在1．76％～2．53％之间，回收率为96．15％-105．09％；UH2在10-400μg·L^-1的范围内线性良好（r=0．9996，n=6），测定的批内变异RSD在3．67％-3．80％之间，批间变异RSD在3．40％～5．74％之间，回收率为101．51％～104．06％。结论：本方法灵敏度高，操作简便易行，为测定体内尿嘧啶和二氢尿嘧啶，进而评价二氢嘧啶脱氢酶活性提供方法学基础。     

Usefulness of uracil loading test for detecting 5-fluorouracil metabolic enzyme deficiencies in humans

Background. We investigated the usefulness of the uracil loading test to detect human carriers of dihydropyrimidine dehydrogenase (DPD) and dihydropyrimidinase (DHPase) deficiencies. Methods. Our subjects consisted of family A (including a patient, a 37-year-old man with a urinary dihydrouracil (DHU) level of 185.4 μmol/mmol creatinine [Cre]); family B (including a patient, a 3-month-old girl with a DHU level of 154.3 μmol/mmol Cre); and ten healthy volunteers. Oral loading tests were performed with 10 mg/kg of uracil to examine changes in the serum and urinary levels of uracil and dihydrouracil in the subjects. Results. The uracil loading test showed the highest levels of urinary DHU 120 min after loading in the mother and father of the patient in family A (52.2 and 65.4 μmol/mmol Cre, respectively) and the mother of the patient in family B (66.4 μmol/mmol Cre). Conclusion. These urinary DHU levels were higher than those in the ten healthy adults, which led us to diagnose these individuals with high DHU levels as human carriers of DHPase deficiencies. Received: July 5, 1999 / Accepted: February 10, 2000     

宁心宝胶囊指纹图谱研究及4种核苷类成分的含量测定

摘 要 目的： 研究宁心宝胶囊的HPLC指纹图谱,并建立同时测定宁心宝胶囊中尿嘧啶、尿苷、腺嘌呤、腺苷含量的方法。方法: 采用Agilent Zorbax SB Aq C₁₈色谱柱（250 mm×4.6 mm,5 μm）,甲醇 0.05 mol·L^-1磷酸二氢钾水溶液为流动相,梯度洗脱,流速为1.0 ml·min^-1,检测波长为212 nm（指纹图谱）、260 nm（含量测定）,柱温为30 ℃。对10 批宁心宝胶囊进行了指纹图谱及含量测定研究,采用中药色谱指纹图谱相似度评价软件进行分析。结果: 建立了宁心宝胶囊的HPLC 指纹图谱,标定了10个共有色谱峰。4种核苷类成分的分离度良好。结论:此方法简便、可靠,指纹图谱结合含量测定可以更好地控制宁心宝胶囊的内在质量。     

稳定剂保护PCR扩增dU-DNA效果研究

目的：了解94℃灭活UDG与不灭活及扩增产物加稳定剂与加稳定剂对DNA杂交效率的影响。方法：采用微孔板DNA杂交技术检测PCR扩增dU-DNA杂交效果。以不加UDG组DNA杂交A值为对照、计算杂交百分率。结果：不加UDG组扩增前后未经94℃灭活处理-20℃保存20h杂交效率为90.293％，加UDG组在4℃、室温、37℃放置20h，杂交效率为20.15％，21.37％，29.37％。加UDG并增加94℃灭活步骤、37℃水浴放置20h，杂交效率为73.57％，78.82％。在此基础上加入稳定剂37℃水浴43h杂交效率达到99.88％-100％。37℃水浴67h杂交效率仍达到86.06％-97.10％。结论：上述结果证实用于抗污染时，扩增前后增设94℃灭活10min可提高杂交效率。加入稳定剂可使杂交A值1.80达到对照组A值1.81水平。提示稳定剂对保护PCR扩增dU-DNA有极其重要作用。     

HPLC法测定血浆中阿糖胞苷及阿糖尿苷浓度

目的：建立同时测定血浆中阿糖胞苷和阿糖尿苷浓度的高效液相色谱法,并用于1例急性非淋巴细胞白血病患儿的血药浓度监测。方法：采用反相高效液相色谱法,色谱柱：XTerra C18（4．6mm&#215;250mm,5μm）;流动相：0．01mol&#183;L^-1的磷酸盐缓冲液（pH=6．0）;检测波长：280nm;流速：0．95mL&#183;min^-1;柱温：30℃。结果：阿糖胞苷血药浓度在0．25～21．74mg&#183;L^-1范围内线性关系良好（r=0．9996）,检测限为0．1mg&#183;L^-1,日内、日间RSD小于5．0％和8．0％;阿糖尿苷血药浓度在0．99～86．96mg&#183;L^-1范围内线性关系良好（r=0．9993）,检测限为0．4mg&#183;L^-1,日内、日间RSD小于5．0％和8．0％。阿糖胞苷及阿糖尿苷平均提取回收率高于96．0％：结论：本方法简单、快速、准确。适用于临床上阿糖胞苷和阿糖尿苷的血药浓度监测及药动学研究．