用固定化大肠埃希氏菌基因工程株转化戊二酰基—7—氨基头孢烷？…

用乙酸纤维将ＧＬ－７－ＡＣＡ酰化酶基因工程菌大肠埃希氏菌Ａ５６／ｐＭＲ ＣＵ－３３４株菌固定化,酶反应的最适温度为５０℃,最适ｐＨ为８．０,与游离细胞一致;固定化细胞对温度和ｐＨ的稳定性比游离细胞提高。用薄板层析分析固定化细胞裂解ＧＬ－７－ＡＣＡ反应液中产物７－ＡＣＡ的含量以及ＧＬ－７－ＡＣＡ的残存量,在ｐＨ８．０．３７℃时ＧＬ－７－ＡＣＡ的转化率达９０％,产物７－ＡＣＡ的能力为１２．３４ｍｇ／（     

药物中间体7-氨基头孢烷酸的制备

药物中间体7-氨基头孢烷酸（7-ACA）的工业化生产国内多局限于化学裂解法，由于该法有某些缺点，因此，本文介绍了环保型的酶法催化生产7-ACA的制备方法。该制备方法以头孢菌素C为起始原料，经固定化D-AOD氧化酶和GL-脱酰酶的作用，酶法裂解制得7-ACA，得到7-ACA的总收率达到74％。实验结果说明该方法是可行的，并且具有重要的现实意义。本文还对影响反应的因素做了进一步的讨论。     

7-氨基头孢霉烷酸工艺研制

实验在头孢锌盐硅烷化过程中，加入了促溶剂N，N-二甲基甲酰胺，使反应周期缩短，两相反应更加充分，脱色过程采用混合炭法，增强了脱色效果。降低了7-ACA色级，从而使7-ACA质量得到了较大幅度的提高。     

毛细管电泳分析7-氨基头孢霉烷酸生产过程

建立了高效毛细管电泳分析法检测7-氨基头孢霉烷酸生产过程.采用未涂层石英毛细管柱,缓冲液为0.01mol/L磷酸盐缓冲液(pH 8.0),运行电压30kV,检测波长254nm.7-氨基头孢霉烷酸、头孢菌素C和戊二酰基-7-氨基头孢霉烷酸的线性范围(mg/ml)分别为0.05～3(r=0.998)、0.1～3.5 (r=0.992)和0.1～5 (r=0.996),迁移时间和峰面积的RSD分别为0.5%、1.0%、0.6%和2.2%、3.0%、2.4%.     

7-氨基-3-乙烯基头孢烷酸的酶解制备

头孢克肟是第三代口服头孢菌素 ,具有高效 ,长效 ,耐酶 ,剂量小等优点。其母核的合成 ,文献 [1]报道一般采用 7-氨基头孢烷酸 (7- ACA)或去乙酰头孢菌素 C钠盐 (DCCNa)为原料。我们初步探索了 C3位引入乙烯基的反应 ,发现难度较大。本文采用 7-苯乙酰胺基 - 3-氯甲基头孢烷酸对甲氧基苄酯(GCLE,2 )为原料 [2 ] ,经 3位 Wittig反应 ,4位脱甲氧苄基及 7位水解等 3步反应制得 7-氨基 - 3-乙烯基头孢烷酸 (7- AVC,1 )。收稿日期 :1999-0 8-16基金项目 :1999年度广州市重点科技攻关计划 (99-Z-119-0 1)作者简介 :许淑文 (196 8) ,女 ,工…     

酶法制备7-氨基-3-乙烯基头孢烷酸

目的：用7-苯乙酰胺基-3-乙烯基头孢烷酸在固定化青霉素酰化酶IPA-750催化下裂解制备7-氨基-3-乙烯基头孢烷酸（7-AVCA）,并获取其最优反应条件。方法：利用正交实验法实验。结论：利用酶法制备7-AV-CA避免了有机溶媒的使用,割除了五氯化磷,同时青霉素酰化酶还能循环使用,具有相当好的社会和经济效益。     

离子对反相高效液相色谱法测定头孢三嗪、7-氨基头孢菌烷酸及7-氨基头孢烯三嗪酸的含量

用四丁基溴化铵离子对试剂，在Ｃ１８色谱柱上同时测定头孢三嗪产品，７氨基头孢菌烷酸原料及７氨基头孢烯三嗪酸中间体的含量。流动相为乙腈—四丁基溴化铵—ｐＨ７０磷酸盐缓冲液—水（３２∶０３２∶４４∶６３６），检测波长２７０ｎｍ。结果表明：７氨基头孢菌烷酸浓度在９３６～２３４μｇ／ｍＬ，７氨基头孢烯三嗪酸浓度在１０４～２６０μｇ／ｍＬ，头孢三嗪浓度在１２９２～３２３μｇ／ｍＬ的范围均存在良好的线性关系，回收率分别在９９７％～９９９％，９９５％～９８％，１０００％～１００４％。     

低温酰化工艺在7-氨基头孢烷酸生产中的应用

目的 将低温酰化工艺应用于7-氨基头孢烷酸的生产，提高7-氨基头孢烷酸质量。方法 降低7-氨基头孢烷酸生产过程中，酰化步骤的反应温度，由(13±1)℃改为(10±1)℃。结果 D-7-ACA和单杂两项杂质，分别由0.30%和0.15%降低至0.25%和0.10%。结论 该工艺改进简单可行，可有效提高7-氨基头孢烷酸质量，适用于产业化生产。     

从青霉素G生产7-氨基脱乙酰氧基头孢烷酸

综述了以青霉素Ｇ钾为原料经氧化,酯化,扩环,水解,脱苯乙酰基等反应制备了７－氨基脱乙酰氧基头孢烷酸的合成工艺。     

发酵法制造7-氨基脱乙酰氧基头孢烷酸技术概述

从棒状链霉菌分离出扩环酶基因，从顶头孢中分离出扩环酶／羟化酶基因和酰化酶基因，以质粒PSELECT为基础的构建插入了这些酶基因的新的质粒，将重组质粒转入产生青霉素的产黄青霉中，在适当条件下培养转化菌株，产生取代的酰基7－氨基脱乙酰氧基头孢烷酸，用适当的酶脱去侧链获得7－氨基脱乙酰氧基头孢烷酸。     

Uptake of phenoxyacetic acid derivatives into Caco-2 cells by the monocarboxylic acid transporters

The uptake mechanism of phenoxyacetic acid (PA) and its chlorine derivatives, 4-chlorophenoxyacetic acid (4-CPA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was investigated using Caco-2 cells. The cells were incubated with PA, 4-CPA, 2,4-D or 2,4,5-T at pH 6.0 and 37 °C. The order of uptake and lipophilicity expressed by n-octanol partition coefficients were PA < 4-CPA < 2,4-D < 2,4,5-T. Incubation at 4 °C or at pH 7.4 significantly decreased these uptake. Furthermore, pretreatment with the protonophore, carbonylcyanide-p-(trifluoromethoxy) phenylhydrazone, or coincubation with benzoic acid, a typical substrate for the proton-linked monocarboxylic acid transporters (MCTs), significantly decreased the uptake of all compounds. The initial uptake rates of all compounds except PA were apparently saturable, suggesting the involvement of a carrier-mediated process. The order of uptake clearance of the compounds was the same as the order of their uptake and lipophilicity. Preloading of cells with benzoic acid significantly increased their uptake except for PA. These results suggest that the uptake of PA, 4-CPA, 2,4-D and 2,4,5-T from the apical membrane of Caco-2 cells is mediated via common MCTs shared, at least in part, with benzoic acid, and the increase in lipophilicity due to the chlor-substitution may increase uptake via the MCTs.     

Uptake of 4-chloro-2-methylphenoxyacetic acid (MCPA) from the apical membrane of Caco-2 cells by the monocarboxylic acid transporter

The cellular uptake mechanism of 4-chloro-2-methylphenoxyacetic acid (MCPA), a phenoxyacetic acid derivative, was investigated using Caco-2 epithelial cells. The cells were incubated with 50 microM MCPA at pH 6.0 and 37 degrees C, and the uptake of MCPA from the apical membranes was measured. The uptake of MCPA was significantly decreased by incubation at low temperature (4 degrees C) and markedly increased by lowering the extracellular pH. Pretreatment with a protonophore, carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (25 microM), or metabolic inhibitors, 2,4-dinitrophenol (1 mM) and sodium azide (10 mM), significantly decreased the uptake of MCPA by 53%, 45% and 48%, respectively. Coincubation of MCPA with 10 mM l-lactic acid or alpha-cyano-4-hydroxycinnamate, which is a substrate or an inhibitor of the monocarboxylic acid transporters (MCTs), significantly decreased the uptake of MCPA by 31% and 20%, respectively, and coincubation with benzoic acid profoundly decreased the uptake by 68%. In contrast, coincubation with succinic acid (a dicarboxylic acid) did not affect the uptake. Kinetic analysis of initial MCPA uptake suggested that MCPA is taken up via a carrier-mediated process [Km=1.37+/-0.15 mM, Vmax=115+/-6 nmol (mg protein)(-1) (3 min)(-1)]. Lineweaver-Burk plots show that benzoic acid competitively inhibits the uptake of MCPA with a Ki value of 4.68 +/-1.76 mM. A trans-stimulation effect on MCPA uptake was found in cells preloaded with benzoic acid. These results suggest that the uptake of MCPA from the apical membrane of Caco-2 cells is mainly mediated by common MCTs along with benzoic acid but also in part by l-lactic acid.     

7-氨基-3-乙烯基头孢烷酸的合成

目的研究7-氨基-3-乙烯基头孢烷酸的合成方法。方法以7-苯乙酰氨基-3-氯甲基头孢烷酸对甲氧苄酯为起始原料，采用化学法和酶解法制得目标化合物，总收率分别达到55．6％和58．5％。结果与结论该工艺原料易得，反应条件温和，收率有所提高，具有工业生产价值。     

盐诱导法高效表达重组人内抑素

内抑素是 1996年底由Ｆｏｌｋｍａｎ研究组首次发现的一种血管生成抑制剂 ,1997年 1月 ,Ｃｅｌｌ杂志报道 :内抑素特异性抑制内皮细胞增殖 ,并对血管生成与肿瘤生长具有很强抑制作用〔3〕。 1997年 11月 ,Ｎａｔｕｒｅ〔6〕 杂志又相继报道了Ｆｏｌｋ ｍａｎ研究组的内抑素研究的新成果 ,内抑素多次使用不引起小鼠副作用 ,说明这是一种反复用药后动物不产生耐药性的药物。由于内抑素具有广谱抗瘤效应、低毒性、可直接针对内皮细胞并且不产生药物耐受性 ,内抑素可能有着较大的临床应用前景。通过基因工程方法大量获取重组人内抑素将对该新型…     

HPLC法测定7-氨基-去乙酰氧基头孢烷酸中有关物质含量

目的建立高效液相色谱法测定7-氨基-3-脱乙酰氧基头孢烷酸(7-ADCA)中有关物质[Δ-2-7-氨基-3-脱乙酰氧基头孢烷酸(Δ-2-7-ADCA)、苯乙酸、扩环酸]。方法色谱柱:Hypersil ODS柱(250 mm×4.6mm,10μm);流速:2.0 mL/min;柱温:30℃。DAD二极管矩阵检测器检测,检测波长:220 nm;进样量:20μL。梯度洗脱:流动相A:0.05 mol/L磷酸盐缓冲液(pH=6.0);流动相B:乙腈。结果Δ-2-7-ADCA、苯乙酸和扩环酸相对于7-ADCA的响应因子为0.56、0.83和1.1;7-ADCA的线性范围为0.59~5.88μg/mL(r=0.999 7),最低检测浓度:0.12μg/mL。结论该方法操作简便、结果准确、稳定,可用于7-ADCA中有关物质含量的测定。     

23-羟基桦木酸对B_(16)细胞系的诱导分化作用

目的评价 2 3 羟基桦木酸对黑色素瘤B16细胞的抑瘤作用。方法以MTT法测定细胞增殖的抑瘤率 ,并以B16细胞形态、黑色素含量、细胞周期变化及体内致瘤能力的测定作为观察指标。结果用 10～ 2 0 μg/ml的2 3 羟基桦木酸作用肿瘤细胞 ,见有不同程度的抑瘤作用 (P <0 .0 0 1)。表现为黑色素生成能力增加 ,细胞生长缓慢。可使B16细胞阻断在G1期 ,肿瘤体积明显缩小。结论 2 3 羟基桦木酸低剂量 (10～ 2 0 μg/ml)对B16细胞有明显的分化诱导作用 ,而对体内外黑色素瘤增殖有明显的抑制作用     

Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells

Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aristolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also participate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-I-mediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells of the human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-I and AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of caspase 3. The proliferation of HK-2 cells was inhibited in a concentration- and time-dependent manner. Cell-cycle analysis revealed that the cells were arrested in the S-phase. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay and the occurrence of a sub-G1 peak. In addition, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These results also suggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway.     

Expression and purification of a recombinant form of human aromatase from Escherichia coli

Aromatase converts androgen to estrogen, a hormone that plays an important role in the development of breast cancer. Aromatase inhibitors have been shown to be a useful endocrine regimen for estrogen-dependent breast cancer. Structure-function studies of aromatase can generate critical structural information for designing highly potent and specific inhibitors. However, aromatase structure-function studies have been hampered by a lack of purified protein. In this report, we describe the construction and expression of a recombinant derivative of human aromatase in Escherichia coli using the pET vector system, and the purification of the enzyme by means of nickel-agarose affinity chromatography. We examined the expression of the full-length, Del-38, C-6xHis-tagged Del-38, and NC-6xHis-tagged Del-38 forms of aromatase. The recombinant aromatase without the first 38 amino acids from the amino-terminus (i.e. Del-38) was found to have a higher activity than the full-length enzyme. Moreover, the addition of two separate hexameric histidine tags at both the amino and the carboxyl-termini (i.e. NC-6xHis-tagged Del-38) increased the binding affinity of the recombinant enzyme to the nickel-agarose. The expressed aromatase (i.e. NC-6xHis-tagged Del-38 aromatase) was eluted from the nickel-agarose with 80 mM EDTA. The total aromatase activity of the 80 mM EDTA-eluted fractions was significantly higher than the detergent-solubilized protein extract, indicating a renaturation process during the nickel-agarose affinity chromatography. Purified aromatase exhibited a single band when analyzed by SDS-PAGE, and activity up to 5.8 nmol/mg/min was obtained using the tritiated water release assay. The K(m) value for androstenedione was determined to be 62+/-24 nM by enzyme kinetic analysis. The recombinant aromatase preparation was also characterized by reduced CO-difference spectral analysis, reaction product extraction assay, and inhibition studies using two aromatase inhibitors (letrozole and anastrozole). The results indicate that the recombinant aromatase from E. coli has catalytic properties identical to those of the enzyme expressed in human tissue and will be very useful for further structure-function studies of aromatase.     

姜黄素诱导乳腺癌MCF-7细胞凋亡

目的 研究姜黄素对人乳腺癌细胞株MCF-7细胞增殖抑制和诱导凋亡作用.方法 MTT法检测姜黄素对MCF-7细胞的增殖抑制作用;流式细胞术(FCM)PI单染检测细胞周期;Annexin V/PI双染法检测细胞凋亡;Western blot法检测Bcl-2和Bax蛋白的表达.结果 姜黄素对MCF-7细胞生长有明显抑制作用,并呈剂量、时间依赖性;姜黄素能使MCF-7细胞阻滞在G1/S期,可以诱导细胞凋亡,Bax蛋白表达上调,而Bcl-2的表达减少.结论 姜黄素对人乳腺癌MCF-7细胞的增殖具有显著的抑制作用并可诱导细胞凋亡.其分子作用机制可能与其上调Bax基因表达水平的同时下调Bcl-2基因表达水平,从而诱导细胞凋亡有关.