In‐depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites

Madagascar periwinkle (Catharanthus roseus) is the major source of terpenoid indole alkaloids, such as vinblastine or vincristine, used as natural drugs against various cancers. In this study, we have extensively analyzed the proteome of cultured C. roseus cells. Comparison of the proteomes of two independent cell lines with different terpenoid indole alkaloid metabolism by 2D‐DIGE revealed 358 proteins that differed quantitatively by at least a twofold average ratio. Of these, 172 were identified by MS; most corresponded to housekeeping proteins. Less abundant proteins were identified by LC separation of tryptic peptides of proteins from one of the lines. We identified 1663 proteins, most of which are housekeeping proteins or involved in primary metabolism. However, 63 enzymes potentially involved in secondary metabolism were also identified, of which 22 are involved in terpenoid indole alkaloid biosynthesis and 16 are predicted transporters putatively involved in secondary metabolite transport. About 30% of the proteins identified have an unclear or unknown function, indicating important gaps in knowledge of plant metabolism. This study is an important step toward elucidating the proteome of C. roseus, which is critical for a better understanding of how this plant synthesizes terpenoid indole alkaloids.     

Complete genome sequence of Slackia heliotrinireducens type strain (RHS 1)

Stackebrandtia nassauensis Labeda and Kroppenstedt (2005) is the type species of the genus Stackebrandtia, and a member of the actinobacterial family Glycomycetaceae. Stackebrandtia currently contains two species, which are differentiated from Glycomyces spp. by cellular fatty acid and menaquinone composition. Strain LLR-40K-21(T) is Gram-positive, aerobic, and nonmotile, with a branched substrate mycelium and on some media an aerial mycelium. The strain was originally isolated from a soil sample collected from a road side in Nassau, Bahamas. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the actinobacterial suborder Glycomycineae. The 6,841,557 bp long single replicon genome with its 6487 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

鸡骨香的萜类成分研究

对传统药用植物鸡骨香的化学成分进行了研究,从鸡骨香根中分离得到4个已知萜类化合物,分别鉴定为:Mallotucin B(Ⅰ)、Cyperenoicacid(Ⅱ)、Ent-spathulenol(Ⅲ)、Cyperenol(Ⅳ)。其中化合物Mallotucin B、Ent-spathulenol和Cyperenol均为首次从鸡骨香根中分离得到。     

3种苦参生物碱对小鼠的毒性作用研究

以小鼠为试验对象,对3种苦参生物碱(氧化苦参碱、槐定碱、苦参碱)的毒性作用进行了系统的分析.结果显示:(1)混合后的苦参生物碱毒性增加,其中苦参碱+槐定碱的增效作用最强,协同毒力指数(c.f)值达59.1,氧化苦参碱+苦参碱混合给药,c.f值为37.3,为增效作用;氧化苦参碱+槐定碱混合给药,c.f值为13.6,为相加作用.(2)血常规检测结果显示,3种苦参生物碱处理组小鼠的白细胞计数(WBC)、血小板总数(PLT)、中粒细胞(GR)较之对照组显著增高;血液生化指标检测结果显示,槐定碱和氧化苦参碱各处理组小鼠的尿素氮(BUN)、肌酐(Cr)水平均极显著高于对照组(P＜0.01),苦参碱各处理组小鼠的丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基酸转移酶(AST)水平比对照组极显著升高(P＜0.01);脏器系数的统计结果显示:槐定碱、氧化苦参碱各处理组小鼠的肾脏系数、心脏系数以及肝脏系数均极显著高于对照组(P＜0.01),苦参碱处理组小鼠的肾脏系数、心脏系数及肺脏系数显著地高于对照组(P＜0.01、P＜0.05、P＜0.05).研究表明,3种苦参生物碱对小鼠肝脏、肾脏有影响,初步确定它们作用的靶器官是肝脏和肾脏.     

Increase in the indole alkaloid production and its excretion into the culture medium by calcium antagonists inCatharanthus roseus hairy roots

Treatment of Catharanthus roseus hairy roots with antagonists, like verapamil and CdCl₂, that block the Ca²⁺ flux across the plasma membrane enhanced the total alkaloid content by 25% and their secretion 10 times. The specific Ca²⁺ chelator, EGTA, stimulated 90% of the total alkaloid secretion. Treatment with inhibitors of intracellular Ca²⁺ movement, like TMB-8 and trapsigargin, enhanced the total alkaloid content by 74% and their secretion into the culture media by 4- to 6-fold. The results suggest that an inhibition of external and internal Ca²⁺ fluxes induces an increase in the indole alkaloid accumulation and secretion in C. roseus hairy roots.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Production of scopolamine by normal root cultures of Brugmansia candida

In this study, the quantification of scopolamine from Brugmansia candida normal roots is described. The roots were obtained and cultured in half salts strength Schenk and Hildebrandt (1/2 SH) liquid medium plus 30 g l^–1 sucrose and supplemented with 3 mg l^–1 of -naphthaleneacetic acid. The quantitative determination of scopolamine was carried out by HPLC. The maximum scopolamine production in Brugmansia candida normal root culture was 3.94±0.01 mg g^–1 dry weight obtained at 24th day of culture.     

Somatic embryogenesis,plant regeneration,and the evaluation of camptothecin content in <Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

Summary Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.     

Synthesis of 9-N-cinchona alkaloid peptide hybrid derivatives: preparation and conformational study of 9-N-acylamino(9-deoxy)cinchona alkaloids

The synthesis and conformational analyses of several 9-N-acylamino(9-deoxy)cinchona alkaloids is presented. Peptides were connected to cinchona alkaloids via a 9-amino group. The synthesis of the new cinchona alkaloid derivatives was performed straightforwardly from 9-amino(9-deoxy)dihydroquinidine via coupling with carboxylic acid chlorides and several dipeptides. Both alkaloid derivatives with the configuration of the corresponding natural product as well as its unnatural epimer were studied. The conformations of the prepared derivatives in solution were determined by NMR spectroscopy. It is shown that the conformation is strongly influenced by the configuration at the 9-position.