南方红豆杉10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶基因的克隆与生物信息学分析

对中国重要药用植物南方红豆杉中的10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶(DBAT)基因进行分离、测序以及生物信息学分析。根据GenBank中已登录的10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶(DBAT)基因cDNA序列设计引物,采用RT-PCR技术从南方红豆杉叶片中克隆了1个DBAT基因Tc-DBAT的全长cDNA。结果显示,Tc-DBAT cDNA含有1个1 323 bp的开放阅读框(open reading frame,ORF),编码440个氨基酸,对应基因组序列含有1个内含子。Tc-DBAT蛋白N端含有5个N-酰基化作用位点和2个保守的N-糖基化作用位点,具有1个保守的B ig-1结构域,多个重要的磷酸化位点以及1个类似钙结合蛋白重复结构。序列同源性比较、系统发生分析以及蛋白质高级结构预测均表明,Tc-DBAT属于转移酶超家族(transferase superfamily),是一个具有乙酰转移酶活性的功能蛋白,能够催化10-去乙酰巴卡亭Ⅲ(10-DAB)10位的乙酰化,从而生成抗癌新药紫杉醇生物合成途径中的最后一个双萜中间体——巴卡亭Ⅲ。有望通过成功克隆和鉴定紫杉醇生物合成途径中的关键酶基因达到提高其半合...     

利用天蓝色链霉菌转化7-木糖紫杉烷的研究

紫杉醇是目前临床治疗癌症的一线化疗药物,资源紧张,价格昂贵。7-木糖-10-去乙酰基紫杉醇(7-XDT)在红豆杉中含量可达紫杉醇的10倍,脱除木糖基后生成的10-脱乙酰紫杉醇(10-DAT)经乙酰化可生成紫杉醇。通过木聚糖平板对不同菌株进行筛选,从52株供试微生物中,发现27株在木聚糖平板上生长良好。经转化实验筛选,发现一株天蓝色链霉菌(Streptomyces coelicolor YUCM 410115)具有转化7-XDT为10-脱乙酰紫杉醇的能力。菌体细胞经破碎离心后,沉淀及上清液均无转化反应出现,而发酵液的硫酸铵沉淀物则可以转化7-XDT生成10-DAT,表明该菌株能产生一种胞外紫杉醇-7-木糖苷酶,发酵液酶活为6 268U。首次发现天蓝色链霉菌能够产生紫杉醇-7-木糖苷酶,为7-XDT转化生产紫杉醇提供了新的酶源。     

大孔树脂吸附分离烟草绿原酸的研究

通过比较8种大孔吸附树脂对烟草绿原酸的吸附分离性能,筛选出适合分离烟草绿原酸的树脂,并对其动态吸附特性进行研究.结果表明,XDA-1树脂对烟草绿原酸不仅吸附量大,而且解吸率高,适合烟草绿原酸的分离富集.该树脂吸附分离烟草绿原酸的工艺参数为:上柱液浓度3.5 mg/mL,pH 3.0,流速3倍柱床体积/h;以6倍柱床体积的40%乙醇进行洗脱,解吸附效果最佳,绿原酸总回收率为80.06%,初步吸附分离得到的产品中绿原酸含量为39.20 g/100 g.     

胡芦巴中两种芪类成分的纯化工艺研究

本文运用高效液相色谱(HPLC)含量分析方法,以胡芦巴种子中土大黄苷和丹叶大黄素为目标化合物,以其总回收率和总含量为指标,采用单因素和响应面分析法,优化了大孔吸附树脂纯化胡芦巴中这两种芪类成分的工艺条件和参数。结果表明,在11种大孔吸附树脂中,HPD300型树脂的纯化效果最佳,最佳纯化工艺条件为:以25.0 mL、pH 5.0的胡芦巴种子粗提液上柱,流速0.5 mL/min,充分吸附后用3 BV去离子水洗柱,然后用27.0 mL 50%乙醇溶液以流速0.6 mL/min进行解吸。此工艺的平均回收率为91.76%;经HPD300树脂纯化后提取物中芪类成分(包括土大黄苷和丹叶大黄素)总含量从15.5%提高到56.94%。     

大孔树脂分离纯化辣椒叶总黄酮的工艺研究

目的:筛选适合分离纯化辣椒叶总黄酮的一种大孔树脂,同时用响应面法进行优化得到最佳纯化工艺。方法:采用热回流法提取辣椒叶总黄酮,以吸附率和解吸率为考察指标,考察6种不同型号的大孔树脂(HPD100、HPD450、HPD600、HPD826、D101、AB-8)对辣椒叶总黄酮的吸附能力与解吸能力,确定最佳树脂。通过动态吸附解吸实验考察此树脂对辣椒叶总黄酮的最佳分离纯化工艺。结果:通过对辣椒叶总黄酮吸附分离性能的分析显示HPD600为最佳树脂,最优工艺为:上样浓度为10 mg/mL,上样量为10 mL,洗脱体积为4 BV,洗脱液流速为4 mL/min,洗脱液pH为7,依次用水、10%、30%乙醇冲洗树脂柱,50%乙醇为洗脱液。纯化后的黄酮纯度435.4 mg/g。结论:该方法简便,操作简单,对辣椒叶总黄酮的纯化效果较好。     

AB-8型树脂对无患子皂苷的动态吸附与解吸性能

采用单因素实验法,以树脂的饱和吸附量、产品的得率及纯度为指标,考察各因素对动态吸附与解吸的影响,优化大孔树脂动态吸附分离无患子皂苷的工艺条件。结果表明,当pH为4.5的无患子皂苷水提液以流速1 mL/min通过高径比为5.4∶1的吸附柱时,AB-8树脂对无患子皂苷的饱和吸附量达568 mg/g;采用1.5 BV的95%乙醇以1 mL/min的流速洗脱吸附后的树脂,产品得率为83.48%、纯度达93.00%;树脂重复使用8次后其吸附解吸性能基本不变。该方法可以用于无患子皂苷的工业化分离提纯。     

产紫杉醇内生真菌枝状枝孢霉MD2的发酵条件优化

[目的]通过优化内生真菌枝状枝孢霉MD2的发酵条件,提高10-去乙酰巴卡亭Ⅲ (10-DAB)和紫杉醇(Taxol)的产量.[方法]采用单因素试验分析不同的培养基初始pH值、培养温度、摇床转速和培养时间对10-DAB和紫杉醇产量的影响,优化枝状枝孢霉MD2的培养条件;以YES为基本培养基,采用单因素试验和正交试验分析添加苯甲酸钠、苯丙氨酸、丝氨酸和甘氨酸4种前体物对10-DAB和紫杉醇产量的影响,优化枝状枝孢霉MD2的培养基组分.[结果]优化后发酵条件为:在初始pH为5.0的300 mL YES培养基中,添加15 mg/L苯甲酸钠、25 mg/L苯丙氨酸、5 mg/L丝氨酸、15 mg/L甘氨酸,接种1 mL枝状枝孢霉MD2的孢子悬液(107-10s个孢子/mL),28.0℃、220 r/min发酵培养12d.在此条件下,枝状枝孢霉MD2的生物量、10-DAB和紫杉醇的产量分别为15.5 g/L、471.5 μg/L和569.5 μg/L,与初始发酵条件相比,分别提高了1.3、3.6和3.4倍.[结论]首次获得了枝状枝孢霉MD2生产10-DAB和紫杉醇的较适摇瓶发酵条件,可为进一步放大发酵培养提供参考.     

紫葳科植物猫爪藤阿克替苷的提取富集工艺

建立UPLC测定猫爪藤Macfadyena unguis-cati阿克替苷含量的方法,采用单因素实验和正交试验优化阿克替苷提取工艺,通过静态、动态吸附与解吸实验筛选适合富集阿克替苷的大孔树脂,并对富集工艺进行研究。UPLC条件:色谱柱HSST3柱(2.1×100 mm,1.8μm);流动相0.1%甲酸(A)-乙腈(B);梯度90%A 5 min,75%A 7.5 min,30%A 9 min,90%A;检测波长310 nm;柱温25℃;流速0.3 mL·min~(-1);保留时间5.21 min。在UPLC条件下阿克替苷在0.25~100 ng进样范围内峰面积与进样量呈良好的线性关系,回归方程为Y=89.002X-4.8275,R=0.9999。单因素实验显示,70%乙醇为最佳提取溶剂。正交试验确定最佳提取条件为70%乙醇,料液比1∶10,提取时间12 h。静态、动态吸附和解吸实验确定HP-20大孔树脂为最佳富集材料。富集工艺为4倍体积15%乙醇洗脱除杂后收集4倍体积40%乙醇洗脱的部分。     

大孔吸附树脂对金银花叶茎藤总黄酮的吸附性能

从金银花叶茎藤中提取总黄酮并用D-101大孔吸附树脂进行纯化,研究了D-101大孔吸附树脂对总黄酮的吸附及解吸附特性。结果表明,D-101树脂对金银花叶茎藤总黄酮分离纯化的最佳工艺参数为:上样液黄酮浓度0.538 mg/mL,静置吸附时间80 min,料液比1∶5(g∶mL),pH 2,流速为2 mL/min,以60 mL 75%的乙醇溶液洗脱,黄酮解吸率为94.5%,纯化后黄酮纯度为84.5%,是粗提液黄酮含量(16.8%)的5倍。金银花叶茎藤总黄酮在D-101树脂上的吸附等温线符合Langmuir等温吸附方程。吸附热力学参数表明吸附过程为自发、放热过程,吸附动力学可用Pseudo-second-order模型较好地拟合,30℃时其表观吸附速率常数为1.034×10-2g/mg.min。     

甜叶菊渣中总黄酮的纯化工艺研究

以甜叶菊渣为原料,采用大孔树脂吸附和溶剂萃取法相结合的方法,得到90％以上纯度的总黄酮.通过对大孔树脂及溶剂萃取法的各影响因素进行研究,确定纯化甜叶菊渣中总黄酮的最佳工艺条件:AB-8型大孔树脂吸附流速为2 mL/min、上样液质量浓度1.5 mg/mL、上样液pH值为3.5、上样量4 BV,解吸液为50％乙醇溶液、解吸量5 BV、解吸流速为1.5 mL/min.优化后的甜叶菊总黄酮平均纯度为50.11％.后经乙酸乙酯在常温条件下萃取5次,得到甜叶菊渣中总黄酮纯度为91.8％.结果表明:通过AB-8型大孔吸附树脂和乙酸乙酯萃取相结合的方法,可以很好地纯化甜叶菊总黄酮.     

Fluorometric high-performance liquid chromatographic analysis of 10-deazaaminopterin, 10-ethyl-10-deazaaminopterin, and known metabolites

The antifolate compounds 10-deazaaminopterin (10-dAM) and 10-ethyl-10-deazaaminopterin (10-EdAM) are therapeutically superior to methotrexate in transplanted murine tumor systems and in human tumor xenografts growing in immunodeficient "nude" mice. The increased therapeutic index of these analogs correlates with their selective uptake, retention, and polyglutamation within neoplastic cells. We have developed a fluorescence high-performance liquid chromatographic assay applicable to 10-dAM, 10-EdAM, their polyglutamate anabolites, and their 7-hydroxy (7-OH) and deglutamate catabolites. The assay is based upon the high native fluorescence of pteridine-containing compounds which contain carbon in the 10 position. The assay employs a reverse-phase C-18 column and an ascending acetonitrile gradient in 50 mM phosphate, pH 7.0. The compounds are extracted from plasma and urine with 95 +/- 7% and 98 +/- 2% recoveries, respectively, using C-18 Sep-Paks. The linear range of the assay is, for 10-dAM, 2-100 nM, and for 10-EdAM, 1-100 nM. Polyglutamated metabolites of [3H]10-EdAM isolated from L1210 cells have been separated by HPLC with identification of five derivatives (Glu 1-5) confirmed by enzymatic peak shift using serum conjugase and by quantitative correlation of fluorescence intensity, radioactivity, and titration inhibition of dihydrofolate reductase. The assay has been used successfully in pharmacokinetic analyses of plasma and urine samples from patients receiving 10-dAM and 10-EdAM. In patients who had received 10-EdAM, 7-OH-10-EdAM, and the deglutamate catabolite were also detected. This HPLC fluorescence assay is superior to the dihydrofolate reductase inhibition and binding assays with regard to specificity and precision; moreover, it can provide a means for simultaneous assay of the physiologically important anabolites and catabolites of these new antifolates.     

Factors affecting the termination of cardiovascular actions of 10, 10-difluoro, 13-dehydroprostacyclin

Experiments were conducted to determine why 10,10-difluoro, 13-dehydroprostacyclin (DF₂-PGI₂) has a long vascular relexant activity but like PGI₂ hads a short duration of effect . DF₂-PGI₂ produced depressor responses in anesthetized dogs which were not affected by nephrectomy suggesting that the kidney was not responsible for the termination of action. DF₂-PGI₂ given intravenously or into the ascending oarta produced depressor responses of a similar magnitude but injection of the same doses into the hepatic portal circulation resulted in a large attenuation of responses. The data suggest hepatitic, but not pulmonary, metabolism of DF₂-PGI₂. Injection or infusion of PGI₂ and DF₂-PGI₂ into the hindlimb circulation caused vasodilation of a similar duration suggesting diffusion from tissue sites as another mechanims of termination of action.     

Oxydromus Grube, 1855 reinstated over Ophiodromus Sars, 1862 (Polychaeta,Hesionidae)

The hesionid polychaete genera Oxydromus Grube, 1855 and Ophiodromus Sars, 1862 have been regarded as synonyms with the former considered as invalid since it was thought to be a junior homonym of Oxydromus Schlegel, 1854. However, Schlegel’s name is an incorrect subsequent spelling for Ocydromus Wagler, 1830 (Aves, Gruiformes, Rallidae) and is not an available name. Consequently, Oxydromus Grube, 1855 must be reinstated for this hesionid polychaete genus. A check-list of valid species of Oxydromus including 30 new combinations is provided.     

Metabolism of d-Glycero-d-Manno-Heptitol, Volemitol, in Polyanthus. Discovery of a Novel Ketose Reductase

Volemitol (d-glycero-d-manno-heptitol, α-sedoheptitol) is an unusual seven-carbon sugar alcohol that fulfills several important physiological functions in certain species of the genus Primula. Using the horticultural hybrid polyanthus (Primula × polyantha) as our model plant, we found that volemitol is the major nonstructural carbohydrate in leaves of all stages of development, with concentrations of up to 50 mg/g fresh weight in source leaves (about 25% of the dry weight), followed by sedoheptulose (d-altro-2-heptulose, 36 mg/g fresh weight), and sucrose (4 mg/g fresh weight). Volemitol was shown by the ethylenediaminetetraacetate-exudation technique to be a prominent phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of the phloem sap carbohydrates, surpassed only by sucrose (63%). Preliminary ¹⁴CO₂ pulse-chase radiolabeling experiments showed that volemitol was a major photosynthetic product, preceded by the structurally related ketose sedoheptulose. Finally, we present evidence for a novel NADPH-dependent ketose reductase, tentatively called sedoheptulose reductase, in volemitol-containing Primula species, and propose it as responsible for the biosynthesis of volemitol in planta. Using enzyme extracts from polyanthus leaves, we determined that sedoheptulose reductase has a pH optimum between 7.0 and 8.0, a very high substrate specificity, and displays saturable concentration dependence for both sedoheptulose (apparent K_m = 21 mm) and NADPH (apparent K_m = 0.4 mm). Our results suggest that volemitol is important in certain Primula species as a photosynthetic product, phloem translocate, and storage carbohydrate.Alditols (sugar alcohols or acyclic polyols) may be chemically described as reduction products of aldose or ketose sugars. The most prevalent plant alditols are the hexitols sorbitol, mannitol, and galactitol. However, as many as 17 different alditols occur naturally in higher plants (for review, see Bieleski, 1982; Lewis, 1984; Loescher and Everard, 1996). The lesser-known alditols are often restricted in their occurrence but still fulfill important functions in those plants where they do occur. Volemitol (Fig. ​(Fig.1) 1) is a good example of a less common but important alditol. This seven-carbon sugar alcohol seems to be confined to certain sections of the genus Primula, so much so that it has been suggested as a useful chemotaxonomical marker (Kremer, 1978). Very little is known about the physiology and metabolism of volemitol in primulas, except that it was an early photosynthetic product in cowslip (Primula veris) and oxslip (Primula elatior) (Kremer, 1978). Figure 1Fischer projections of volemitol and its four structurally related seven-carbon sugars. Nomenclature follows that of Collins (1987); trivial names are underlined.The physiological roles of alditols are manifold and largely resemble those of disaccharides and oligosaccharides. They include photosynthetic assimilation, translocation and storage of carbon, and reducing power, as well as protection against different types of stresses (for review, see Bieleski, 1982; Lewis, 1984; Loescher and Everard, 1996; Stoop et al., 1996). The biosynthetic pathways of the hexitols sorbitol (glucitol), mannitol, galactitol (dulcitol), and the pentitol ribitol have been established in higher plants. They generally use NADPH as a hydrogen donor and aldose phosphate as a hydrogen acceptor, in concert with the corresponding phosphatases. One exception might be galactitol, which was suggested to be formed directly from unphosphorylated Gal (and NADPH) (Negm, 1986). Although all foliar alditols are thought to be phloem-mobile (Lewis, 1984), this has only been demonstrated for sorbitol, mannitol, and galactitol (Zimmermann and Ziegler, 1975; Davis and Loescher, 1990; Moing et al., 1992; Flora and Madore, 1993).To expand our knowledge of alditol metabolism in higher plants beyond that of hexitols, we studied the carbohydrate metabolism of polyanthus (Primula × polyantha). This popular horticultural hybrid of primrose (Primula vulgaris), oxlip, and cowslip (Mabberley, 1997) was chosen because preliminary experiments showed that its volemitol content is very high, similar to that of the wild-type species, and because it may be easily grown both outdoors and indoors.We give a general overview on volemitol metabolism in polyanthus with special emphasis on the role of volemitol in plant development and phloem transport. We also report on a novel enzyme, a NADPH-dependent ketose reductase, which forms volemitol by the reduction of sedoheptulose.     

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl⁻ and K⁺. The postshrinking volume recovery is achieved by K⁺ and Cl⁻ influxes, with contribution by the proton motive force. External Ca²⁺ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.