6种川产姜黄属药用植物叶绿体trnK基因序列变异分析及其分子鉴定

目的建立6种川产姜黄属(Curcuma)药用植物快速简单的分子鉴定方法.方法采用叶绿体赖氨酸tRNA基因(trnK)测序与序列变异分析方法.结果 6种姜黄属药用植物(包括姜黄C. longa、莪术C. phaeocaulis、川郁金C. sichuanensis、川郁金C. chuanyujin、川黄姜C. chuanhuangjiang、川莪术C. chuanezhu)完整trnK基因长度在2699～2705 bp.序列可变区包括matK基因编码区和trnK外显子与matK内含子之间区域,共有6个单核苷酸多态性(SNPs)位点、1个9-bp的缺失重复序列和2个4-bp、14-bp插入重复序列.结论 trnK基因序列可变位点可以作为6种川产姜黄属药用植物快速简单的分子鉴定标记,并为它们之间种的归并提供了分子依据.     

Molecular analysis of medicinally-used Chinese and Japanese Curcuma based on 18S rRNA gene and trnK gene sequences.

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. In order to develop an ultimate identification, molecular analysis based on 18S rRNA gene and trnK gene sequences were performed on 6 Curcuma species used medicinally in China and Japan. The 18S rRNA gene sequences were found to be of 1810 bps in length. In comparison with the common sequence of C. longa, C. phaeocaulis, C. wenyujin and C. aromatica, that of C. kwangsiensis had one base substitution, and the same base difference was observed between the Chinese and the Japanese populations of C. zedoaria. The trnK gene sequences were found to span 2698-2705 bps. There were base substitutions, small deletions or insertions at some sites between the trnK coding region and matK region among each species. Based on the base substitutions, C. zedoaria and C. kwangsiensis specimens were divided into two groups, respectively. An identical sequence was detected in C. phaeocaulis and in the Chinese population of C. zedoaria, as well as in the Japanese population of C. zedoaria and in one group of C. kwangsiensis with a purple-colored band in leaves. New taxonomic information to be used for authenticating Curcuma drugs was obtained.     

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.     

A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity

To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01). The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol). This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype. Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies.     

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.     

Authentication of Curcuma species (Zingiberaceae) based on nuclear 18S rDNA and plastid trnK sequences

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.     

Serial analysis of gene expression: from gene discovery to target identification

Serial Analysis of Gene Expression (SAGE) is a sequence-based genomics tool that features comprehensive gene discovery and quantitative gene expression capabilities. As an 'open' system, SAGE can reveal which genes are expressed and their level of expression rather than merely quantifying the expression level of a predetermined, and presently incomplete, set of genes as carried out by 'closed' system gene expression profiling platforms such as microarrays. These distinguishing attributes enable SAGE to be used as a primary discovery engine that can characterize human disease at the molecular level while illuminating potential targets and markers for therapeutic and diagnostic development, respectively.     

Morphological, genetic, and chemical polymorphism of Curcuma kwangsiensis

Previously, Chinese Gajutsu available in Japan was identified, from the chloroplast trnK gene sequence, to be the rhizomes of Curcuma phaeocaulis and two genotypes of C. kwangsiensis. Although we defined the two genotypes, the pl and gl types, on the basis of the nucleotide difference, their external features did not correspond to the two phenotypes described in the literature. In this paper, to investigate the relationship between genotype and phenotype of C. kwangsiensis, a field investigation was carried out in its main cultivation areas of Guangxi Zhuangzu Autonomous Region and Guangdong Province, China, and sequence analysis of the trnK gene and single-nucleotide polymorphism (SNP) analysis of the nuclear 18S rRNA gene were performed on the collected specimens. Four genotypes of C. kwangsiensis were recognized from the combined 18S rRNA gene–trnK gene sequences: homozygote-K(gl)Wtk type, homozygote-K(pl)Ztk type, heterozygote-K(gl)Wtk type, and heterozygote-Ltk type. Among the four genotypes, C. kwangsiensis in a field used for cultivation of Gajutsu was of heterozygote-K(gl)Wtk type. Formation of a heterozygote in the 18S rRNA gene might be a result of crossbreeding of C. kwangsiensis with several Curcuma species which had cytosine at nucleotide position 234. GC analysis of the rhizomes revealed that C. kwangsiensis was characterized by camphor and β-elemene, and by detecting additional components such as curdione and curcumenol Curcuma species involved in the formation of the heterozygote might be speculated upon.     

龙胆目查尔酮合成酶的生物信息学分析

目的运用生物信息学方法分析GenBank中登录的4种龙胆目植物的5个查尔酮合成酶基因(CHS)。方法分别用ExPASy ProtParam Tool、Prot Scale和Mega等软件,对5个基因的理化性质、跨膜结构、蛋白质二级结构和三级结构及结构域进行了分析和预测,并利用距离矩阵方法中的邻接法构建CHS系统发育树。结果与结论龙胆目CHS基因编码区(CDS)的全长均为1 170 bp,大约编码389个氨基酸;其中只有华北白前的氨基酸序列预测结果是不稳定蛋白质,另外4个均为稳定的蛋白质;5个都是疏水性蛋白质;二级结构中主要结构元件是α螺旋和不规则卷曲;都具有查尔酮合成酶蛋白典型的结构域;利用同源建模的方法预测了三维结构,与预测的二级结构结果相一致;均无信号肽和跨膜结构。根据CHS蛋白的氨基酸序列对龙胆目及另外62种植物进行了系统分析,四种植物的氨基酸序列聚在一起,它们与唇形目和茄目的同源关系较近,得到的这些相关信息可以为之后进一步的研究奠定基础。     

Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences

Chloroplast trnK gene and nuclear 18S rRNA gene sequences of 13 Panax taxa, collected mainly from Sino-Japanese floristic region, were investigated in order to construct phylogenetic relationship and to assist taxonomic delimitation within this genus. The length of trnK gene sequence varied from 2537 bp to 2573 bp according to the taxa, whereas matK gene sequences, embedded in the intron of trnK gene, were of 1512 bp in all taxa. Species-specific trnK/ matK sequence provided much insight into phylogeny and taxonomy of this genus. 18S rRNA gene sequences were of 1808 or 1809 bps in length, only 9 types of 18S rRNA sequences were observed among 13 taxa. Parsimony and neighbor-joining analyses of the combined data sets of trnK-18S rRNA gene sequences yielded a well-resolved phylogeny within genus Panax, where three main clades were indicated. P. pseudoginseng and P. stipuleanatus formed a sister group located at a basal position in the phylogenetic tree, which suggested the relatively primitive position of these two species. Monophyly of P. ginseng, P. japonicus (Japan) and P. quinquefolius, which are distributed in northern parts of Asia or America, was well supported (Northern Clade). The remaining taxa distributed in southern parts of Asia formed a relatively large clade (Southern Clade). The taxonomic debated taxa traditionally treated as subspecies or varieties of P. japonicus or P. pseudoginseng showed various nucleotide sequences, but all fell into one cluster. It might suggest these taxa are differentiated from a common ancestor and are in a period of high variation, which is revealed not only on morphological appearance, but also on molecular divergence. By comparing trnK and 18S rRNA gene sequences among 13 Panax taxa, a set of valuable molecular evidences for identification of Ginseng drugs was obtained.     

Pharmaceutical target identification by gene expression analysis

The majority of newly-identified genes in the human genome show no significant sequence similarity to genes whose function is known, so they are not easily recognized as potential drug targets. Expression analysis is an alternative method to suggest possible functions of genes. We review statistical methods for gene expression analysis to identify potential pharmaceutical targets. Specifically, we illustrate the analysis of differential gene expression (using discriminant analysis, t-tests, and analysis of variance) and co-expression (using correlation, clustering, and chi-square). We present an example of the use of expression analysis to identify co-expressed cardiomyopathy-associated genes.     

现代技术在莪术研究中的应用

莪术在提取、药理学研究、质量控制、种属鉴定中广泛运用了分子生物学技术等现代技术，文章介绍了目前莪术的最新研究技术。     

Infrared identification of barbiturates with particular reference to the occurrence of polymorphism

Infrared spectra of twelve substituted barbituric acids, in a total of 34 polymorphic forms, have been compared. Comparison of a sample spectrum with that of an authentic specimen provides a reliable means of identification, provided that both are in the same crystalline form. To ensure consistent production of a single form a specific treatment is recommended for each substance exhibiting polymorphism.     

Rapid identification of Curcuma longa and C. aromatica by LAMP

We have developed a novel method for the identification of Curcuma longa and C. aromatica called "loop-mediated isothermal amplification (LAMP)," based on trnK gene sequences. LAMP employs four primers that recognize six regions on the target DNA. Cycling elongation was initiated when the four primers were annealed to the target DNA. Amplifications were detected by measuring turbidity due to the formation of magnesium pyrophosphate. We designed allele-specific primer sets for C. longa and C. aromatica, respectively. LAMP using a primer set for C. longa and total DNA extracted from C. longa rhizome (0.5-10.0 ng) as template was detected up to 70 min. On the other hand, in the reaction using a primer set for C. longa and total DNA from C. aromatica as template, no amplifications were detected. The same tendency could be seen in the reactions using a set of primers for C. aromatica. LAMP enabled not only identification but also detection with high specificity. This rapid, specific, sensitive, and convenient method is expected to be applicable to the identification of the botanical origin of commercially available herbal products.     

毛郁金化学成分的分离与鉴定

目的对毛郁金的化学成分进行研究。方法采用硅胶柱色谱、氧化铝柱色谱、ODS柱色谱和制备高效液相色谱等方法分离纯化毛郁金的化学成分,通过波谱数据分析确定化合物的结构。结果从毛郁金乙酸乙酯萃取物中分离得到10个化合物,分别鉴定为莪术二酮(curdione,1)、新莪术二酮(neocurdione,2)、吉马酮-4,5-环氧化物(germacrone-4,5-epoxide,3)、莪术二酮-1,10-环氧化物(curdione-1,10-epoxide,4)、gajutsulactone A(5)、莪术双环烯酮(curcumenone,6)、polydactin B(7)、4,5-seco-4,5-dioxo-guaia-1(10),11-diene(8)、2-endo-hydroxy-1,8-cineole(9)、邻苯二甲酸二丁酯(dibutylphthalate,10)。结论化合物3、5、7-10为首次从该植物中分离得到,其中化合物7-10为首次从姜黄属植物中分离得到。     

姜黄化学成分的分离与鉴定

目的研究姜黄的化学成分。方法采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱法和HPLC法等方法对姜黄体积分数为95%的乙醇提取物的乙酸乙酯层的化学成分进行分离与纯化,并根据理化性质、NMR、MS等波谱数据鉴定化合物的结构。结果分离得到6个化合物,分别鉴定为:芳姜黄酮(ar-tumerone,1)、14-羟基芳姜黄酮(6-(4-hydroxy methyl phenyl)-2-methyl-hept-2-ene-4-one,2)、isobisabolone A(3)、姜黄酮(turmeronol,4)、2-methyl-6-(4-hydroxyphenyl)-2-hepten-4-one(5)、2-methyl-6-(2-cyclohexen-4-one)-2-hepten-4-one(6)。结论化合物2为新的天然产物,化合物6为首次从姜黄属中分离得到。     

Application of 12S rRNA gene for the identification of animal-derived drugs

PURPOSE. Animal-derived drugs are the major source of biological products and traditional medicine, but they are often difficult to identify, causing confusion in the clinical application. Among these medicinal animals, a number of animal species are endangered, leading to the destruction of biodiversity. The identification of animal-derived drugs and their alternatives would be a first step toward biodiversity conservation and safe medication. Until now, no effective method for identifying animal-derived drugs has been demonstrated; DNA-based species identification presents a brand-new technique. METHODS. We designed primers to amplify a 523-bp fragment of 12S rRNA and generated sequences for 13 individuals within six medicinal animal species. We examined the efficiency of species recognition based on this sequence, and we also tested the taxonomic affiliations against the GenBank database. RESULTS. All the tested drugs were identified successfully, and a visible gap was found between the inter-specific and intra-specific variation. We further demonstrated the importance of data exploration in DNA-based species identification practice by examining the sequence characteristics of relative genera in GenBank. This region of the 12S rRNA gene had a 100% success rate of species recognition within the six medicinal animal species. CONCLUSIONS. We propose that the 12S rRNA locus might be universal for identifying animal-derived drugs and their adulterants. The development of 12S rRNA for indentifying animal-derived drugs that share a common gene target would contribute significantly to the clinical application of animal-derived drugs and the conservation of medicinal animal species. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.     

CD40基因多态性与急性冠状动脉综合征的相关性研究

目的研究CD40基因多态性位点CD40-E1SNP(-1C/T)与急性冠状动脉综合征(ACS)的关系及CD40-CD40L信号通路在ACS中所起的作用。方法应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法检测166例经冠状动脉造影确诊的急性心肌梗死(AMI)患者74例和不稳定心绞痛(UAP)患者92例以及77名冠状动脉造影阴性的健康人群中CD40-E1SNP(-1C/T)的基因型和等位基因分布频率,结合血清炎性标记物可溶性CD40配体(sCD40L)、基质金属蛋白酶(MMP)-2分析CD40-CD40L信号通路在ACS中所起的作用。结果 CD40-1位点C等位基因的分布频率在AMI组、UAP组及健康对照组中分别为65.5%、54.3%、45.5%,差异有统计学意义(P<0.05)。CC基因型在AMI组、UAP组及健康对照组中的分布频率分别为41.9%、28.3%、19.5%,差异有统计学意义(P<0.05)。与对照组相比,AMI组与UAP组血清中sCD40L、MMP-2含量均增高,同时AMI组高于UAP组,差异均有统计学意义(P<0.05),且3组间sCD40L与MMP-2含量呈线性相关,MMP-2含量随着sCD40L含量的升高而呈上升趋势,具有统计学意义(P<0.05)。结论 CD40基因多态性位点CD40-E1SNP与ACS有相关性,C等位基因可能是ACS的易感基因,且是AMI的易发因素;血清中sCD40L、MMP-2含量水平升高是动脉粥样硬化斑块不稳定的标志,可作为心血管危险事件的预测因子;CD40-CD40L系统在ACS的发生发展中起着重要的作用。     

Infrared identification of pharmaceutically important sulphonamides with particular reference to the occurrence of polymorphism

The infrared absorption spectra of sulphonamides, when compared with the spectra of Authentic Specimens, provide a simple and complete means of identification, provided the effects of polymorphism are excluded. Of 18 substances examined, twelve showed evidence of polymorphism. Limited solubility prevents the use of solution spectra, and specified solvent treatments, details of which are given, may therefore be necessary with these substances to ensure reproducible spectra.