Phylogeny of Astragalus in China: molecular evidence from the DNA sequences of 5S rRNA spacer, ITS, and 18S rRNA

Radix Astragali (root of Astragalus; Huangqi) is a traditional Chinese medicine commonly used as an immunostimulant, hepatoprotective, diuretic, antidiabetic, analgesic, expectorant, and sedative drug. Although the species of Radix Astragali have been defined as Astragalus membranaceus and A. membranaceus var. mongholicus in Pharmacopoeia of China, their taxonomy remains controversial. The phylogenetic relationships among 10 Astragalus taxa, which are commonly found in China including A. membranaceus, A. membranaceus var. mongholicus, Astragalus propinquus, Astragalus lepsensis, Astragalus aksuensis, Astragalus hoantchy, Astragalus hoantchy subsp. dshimensis,Astragalus lehmannianus, Astragalus sieversianus, and Astragalus austrosibiricus, were determined using the DNA sequences of the 5S ribosomal RNA (5S rRNA) spacer, internal transcribed spacer region (ITS), and 18S rRNA coding region. The 5S rRNA spacer, ITS, and 18S rRNA, amplified by polymerase chain reaction from the isolated genomic DNAs, were sequenced. By using neighbor-joining and maximum parsimony analyses, phylogenetic trees were mapped by their sequence diversity. A. membranaceus and A. membranaceus var. mongholicus shared the greatest sequence homology. In addition, A. propinquus shared a closer relationship with A. membranaceus and A. membranaceus var. mongholicus, while other Astragalus species were less closely related. This is the first paper to show the phylogenetic relationship of Astragalus species related to Radix Astragali in China by the molecular genetic approach.     

Development of a 16S rRNA microarray approach for the monitoring of rhizosphere Pseudomonas populations associated with the decline of take-all disease of wheat

So far, the analysis of microbial populations associated with wheat monocropping-induced decline of take-all disease (Gaeumannomyces graminis var. tritici) has focused mainly on culturable biocontrol pseudomonads. The objective of this study was to develop a taxonomic rrs (16S rRNA gene) microarray to assess the changes in Pseudomonas populations taking place during take-all decline. The microarray contains 12 probes for five Pseudomonas phylogenetic clusters chosen because they include well-known plant-beneficial pseudomonads. Four of the clusters are within the ‘Pseudomonas fluorescens’ species complex. PCR primers were selected to target these five clusters, and they were validated using 53 pseudomonads belonging or not to these clusters. Microarray analysis of the pseudomonads enabled discrimination between strains from several Pseudomonas clusters. Rhizosphere samples were collected from field plots grown with wheat for 1 (low level of take-all disease), 5 (high level of disease) or 10 years (low level of disease, suppressiveness reached). Microarray data could distinguish Pseudomonas populations from some of the wheat plants grown in the same plot. When comparing treatments, there was a difference between years 1 and 10. Cloning–sequencing of rrs enabled to define more precisely this difference by identifying two major Pseudomonas populations, one associated with year 1 and the other with year 10 (disease suppressiveness), which represent new clades within the ‘P. fluorescens’ complex. These populations may be useful as soil quality indicators. In conclusion, the combination of microarray and cloning–sequencing approaches highlighted changes in the prevalence of two major Pseudomonas populations, giving new insights on the dynamics of root-associated pseudomonads during take-all decline.     

The 5S rRNA gene sequence variation in wheats and some polyploid wheat progenitors (Poaceae: Triticeae)

We have cloned and sequenced 115 repeat units of the 5S rDNA genes and spacers from wheat (Triticum) and the polyploid wheat progenitor, Aegilops, and analyzed them together with sequences available in GenBank® (National Center for Biotechnology Information, NCBI, NLM, NIH, Bethesda, Maryland, USA). We were able to assort the sequences into nine orthologous groups which we call unit classes. The following unit classes were assigned to haplomes, and labeled accordingly: long A1, short A1, short A2, long G1, short G1, long D1, short D1, long S1 and short S1. The AA-genome, DD-genome and SS-genome species were each found to contain a long and a short class. The AAGG-genome species T. timopheevii and the AAA^tA^tGG-genome species T. zhukovskyi, both contain the long A1, long G1 and short G1 unit classes. The AABB-genome species T. turgidum consists of the short A2, a unit class not yet found in T. monococcum, and the long S1 unit class found in the species of Aegilops section Sitopsis. The bread wheat AABBDD-genome contains the long A1, short A2, long D1, long S1 and short G1 unit classes. The presence of the long S1, also demonstrated to occur in both T. turgidum and T. aestivum, supports the hypothesis that the progenitor of the B-haplome in wheat originated in Aegilops section Sitopsis. The presence of the short G1 unit class, i.e. the G-haplome in bread wheat, is unexpected. These new findings are discussed in the light of published findings, especially those relating to 5S DNA loci and evolutionary hypotheses.     

Analysis of 18S rRNA gene of Octostigma sinensis (Projapygoidea: Octostigmatidae) supports the monophyly of Diplura

The phylogenetic position of Diplura within Hexapoda has been controversial. There are three major lineages in Diplura: Campodeoidea, Projapygoidea, and Japygoidea. However, most of the previous studies were restricted to Campodeoidea and Japygoidea. Until now, only preliminary morphological study on Projapygoidea was reported, and no sequence data from Projapygoidea was available. The main aim of the present study was to investigate the phylogenetic position of Octostigmatidae, one of the three families of Projapygoidea, in Diplura and to test if Diplura are monophyletic. The complete 18S rRNA gene sequences of Octostigma sinensis (Projapygoidea: Octostigmatidae) from subtropical China, together with representative species of Campodeoidea and Japygoidea, and several species of Protura and Collembola were analyzed. The phylogenetic trees were obtained by different methods (neighbor-joining, maximum parsimony, and maximum likelihood) with a chelicerate species as outgroup. Our results suggested that Octostigma was closer to the genus Parajapyx (Japygoidea: Parajapygidae) than to the representative genus of Campodeidae (Campodeoidea). All phylogenetic trees supported the monophyly of Diplura.     

The origin of the A genome donor of wheats (Triticum: Poaceae) – a perspective based on the sequence variation of the 5S DNA gene units

In a prior study on the haplomes of wheat using the 5S rRNA gene we assigned the long A1 and short A1 unit classes to the A haplome in the diploid T. monococcum. The short A1 unit class is absent in the tetraploids T. turgidum and T. timopheevii and in the hexaploid T. aestivum, although present in the hexaploid T. zhukovskyi. Both T. turgidum and T. aestivum contained a different 5S DNA unit class labeled the short A2.The purpose of this paper was to study the short A2 units in the two diploid species to shed light on the theory that the A haplome donor of T. turgidum and T. aestivum was T. urartu. Fifty eight clones were obtained from 12 accessions, sequenced and analyzed. As expected T. baeoticum, which is often classified as a subspecies of T. monococcum, contained the long A1 and the short A1 5S DNA units. Unexpectedly, T. urartu had the long A1 and the short G1 unit classes instead and other units not found so far in Triticum. These findings support the hypothesis that the donor of the A genome in T. zhukovskyi was T. monococcum, as identified by the short A1 units. However, the short A1 units are absent in T. timopheevii, also a carrier of the A genome. The short G1 units found in T. urartu also identify it as a possible donor of the G genome to T. timopheevii. The short G1 units were also found in T. aestivum in our prior study. The long G1 unit class was not found in T. urartu but reported from T. timopheevii and T. zhukovskyi. The implications of these and related findings on the evolution of wheats are discussed.     

A phylogenetic microarray targeting 16S rRNA genes from the bacterial division Acidobacteria reveals a lineage-specific distribution in a soil clay fraction

We designed an oligonucleotide microarray using probe sequences based upon a phylogenetic analysis of 16S rRNA genes recovered from members of the bacterial division Acidobacteria. A total of 42,194 oligonucleotide probes targeting members of the Acidobacteria division at multiple phylogenetic levels were included on a high-density microarray. Positive control hybridizations revealed a linear relationship between hybridization signal and template concentration, and a substantial decrease in non-specific hybridization was achieved through the addition of 2.5 M betaine to the hybridization buffer. A mean hybridization signal value was calculated for each Acidobacteria lineage, with the resultant lineage-specific hybridization data revealing strong predictive value for the positive control hybridizations. The Acidobacteria phylochip was then used to evaluate Acidobacteria rRNA genes from a Wisconsin soil and within a soil clay fraction. The Acidobacteria hybridization profile revealed the predominance of Acidobacteria subdivisions four and six, and also suggested a decrease in the abundance of subdivision six relative to subdivision four in the soil clay fraction. The change in relative abundance of these subdivisions in a soil clay fraction was supported by data from quantitative PCR. These results support the utility of a phylogenetic microarray in revealing changes in microbial population-level distributions in a complex soil microbial assemblage.     

丝瓜18S rRNA基因克隆及其作为内参基因的应用

选择合适的内参基因是提高实时荧光定量PCR分析(q RT-PCR)准确性的重要条件。18S r RNA基因表达范围广、表达量恒定,常作为内参基因应用于实时荧光定量PCR中。为了获得丝瓜18S r RNA基因,并设计合适的荧光定量PCR内参引物,解决丝瓜实时荧光定量PCR检测中无内参基因的现状,通过PCR和序列测定,首次克隆到了丝瓜的18S r RNA基因序列,其长度为1 862 bp,Gen Bank登录号为KM656452。在此基础上设计1对荧光定量PCR引物,该引物特异性强,扩增效率高,在丝瓜各生长发育阶段及各种非生物胁迫条件下均能稳定表达,适合在丝瓜基因表达研究中作为内参基因。该研究结果可为开展丝瓜重要功能基因的表达模式和调控机制的研究奠定基础。     

PCR结合表型鉴定对超高压处理后的冷藏带鱼细菌菌相分析

为了研究超高压处理对带鱼微生物菌群组成的影响,该文通过形态学特征、生理生化鉴定、16S r RNA序列分析鉴定及系统发育树的建立,分别分析290 MPa、6 min超高压处理前后于4℃冷藏12 d内的带鱼菌相变化,最终分离筛选到24株不同特征的纯化菌株。结果显示,带鱼初始菌相中出现的布式假单胞菌(Pseudomonas brenneri)、黄褐假单胞菌(Pseudomonas fulva)、粪嗜冷杆菌(Psychrobacter faecalis)菌株,经超高压处理后的样品中未能筛选到;波罗的海希瓦氏菌(Shewanella baltica)、隆德假单胞菌(Pseudomonas lundensis)、嗜根寡养单胞菌(Stenotrophomonas rhizophila)、表皮葡萄球菌(Staphylococcus epidermidis)、氧化微杆菌(Microbacterium oxydans)等微生物在超高压处理后的贮藏期间数量逐渐减少至消失;另有一些微生物在贮藏期间逐渐恢复生长,如Rhizobium larrymoorei、Microbacterium halimionae、溶酪大球菌(Macrococcus caseolyticus),而奥斯陆莫拉菌(Moraxella osloensis)、藤黄微球菌(Kocuria rhizophila)、产乳酸菌素的肉杆菌(Carnobacterium maltaromaticum)、西宫皮肤球菌(Dermacoccus nishinomiyaensis)等受超高压的影响较小,尤其是产乳酸菌素的肉杆菌(Carnobacterium maltaromaticum)占好氧菌和厌氧菌菌落总数的比例均较高;Leucobacter aerolatus、成团泛菌(Pantoea agglomerans)、结肠炎耶尔森杆菌palearctica亚种(Yersinia enterocolitica subsp.palearctica)、Chryseobacterium vrystaatense、鼠李糖短杆菌(Brachybacterium rhamnosum)在贮藏末期出现。从带鱼冷藏过程中细菌的组成与变化分析可见,超高压处理对革兰氏阴性菌的抑菌效果较好,而革兰氏阳性菌对超高压处理的耐受性较强。在超高压技术的影响下,致腐败能力较强的微生物被抑制,腐败能力稍弱的微生物成为优势菌,这可能是超高压技术能够有效延长带鱼货架期的因素之一。     

16S rRNA基因高通量测序分析牛粪发酵细菌多样性

将养殖粪便进行资源化处理,尤其是将粪便堆肥发酵后变为生物肥料还田,具有重要的经济、社会和生态效益。之前关于细菌在堆肥过程中的研究,大部分采用实验室培养、分离、鉴定的方法,由于受培养方式的限制,仅能分析粪肥中有限的细菌类别。16S r RNA基因作为生物物种的特征核酸序列,被认为是最适于细菌系统发育和分类鉴定研究的指标。本研究使用16S r RNA基因高通量测序技术,分析了牛粪自然发酵与添加益生菌剂发酵过程中细菌种群的多样性变化。结果表明,1)新鲜牛粪、自然发酵1个月、自然发酵6个月的牛粪中细菌种群并没有明显的变化规律,说明自然发酵过程主要依赖于新鲜牛粪中携带的细菌种群;2)添加益生菌发酵后,细菌种群明显不同于不自然发酵过程中的细菌种群,其中变形菌门(Proteobacteria)细菌显著增加,而厚壁菌门(Firmicutes)细菌显著减少,说明益生菌剂能够显著改变堆肥过程中的细菌种群。本研究对于理解牛粪堆肥过程、提高堆肥效果,以及新型堆肥益生菌剂的开发都具有重要意义。     

Identification of marker genes for intestinal immunomodulating effect of a fructooligosaccharide by DNA microarray analysis

Prebiotic fructooligosaccharides are noted for their intestinal immunodulating effects, and the identification of markers for the effects is a matter of great concern. This study aimed to identify marker genes for physiological effects of a particular fructooligosaccharide (FOS) on a host animal and also to define the target of its function in the small intestine. DNA microarray technology was used to screen candidate marker genes, and comprehensive changes in gene expressions in the ileum of mice fed with FOS were investigated. One of the major physiological effects of FOS was intestinal immunomodulation. Marker genes were then identified for major histocompatibility complex classes I and II, interferon, and phosphatidylinositol metabolites. Also, the ileum was segmented into Peyer's patch (PP) and the other ileal organ (DeltaPP), and these were analyzed by quantitative RT-PCR method, with the result that the site for recognizing the FOS function was the DeltaPP rather than the PP. This is the first paper showing the markers for the physiological effects of FOS in the small intestine at gene expression level. Applying these marker genes would make it possible to clarify the mechanisms of how the administration of dietary FOS and associated changes in the intestinal environment are recognized by host organisms as well as how its immunomodulating effects are expressed in the body.     

Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996     

基于3S技术联合的农田墒情远程监测系统开发

农田墒情信息是现代农业实施精准施肥、精确灌溉的重要科学依据。为了实现快速准确地采集墒情信息,研究开发了基于3S（GPS/GIS/GPRS）技术联合的农田墒情远程监测系统。该系统主要由农田信息监测网络节点和远程服务器组成,在小范围内由传感器节点基于ZigBee通讯协议组成无线传感器网络,在大尺度上通过网关节点集成GPS网络,利用GSM/GPRS网络实现与Internet的信息交互,完成了墒情数据的自动采集、无线传输和准确定位。设计了太阳能自供电的长寿命无线传感器节点和网关节点,开发了服务器端农田墒情信息管理系统软件,实现了Web方式下的参数远程设置和信息实时监测。该系统的设计开发为农田墒情信息监测和分析决策提供了有效的工具。     

Flexible microarray construction and fast DNA hybridization conducted on a microfluidic chip for greenhouse plant fungal pathogen detection

This study employed a microfluidic method in which probe creation does not require pin-spotting and fast hybridization is conducted on the same microarray chip for the detection of three greenhouse pathogens ( Botrytis cinerea, Didymella bryoniae, and Botrytis squamosa). In this method, 16 oligonucleotide probe line arrays were created on a glass substrate by a microfluidic printing method. Then, low amounts of the DNA samples (1 fmol of oligonucelotides or 1.4 ng of PCR products) were introduced into the microchannels that were orthogonal to these probe lines. The hybridizations of 16 samples (21-mer complementary oligonuleotides and approximately 260 bp PCR products) were fulfilled at the channel-probe line intersections and in a short time (minutes). The optimization of probe immobilization and sample hybridization are described in detail. The method successfully detected and discriminated between two 260 bp PCR products with a one-base-pair difference from closely related greenhouse plant fungal pathogens (B. cinerea and B. squamosa).     

The characterization and quantification of methanotrophic bacterial populations in constructed wetland sediments using PCR targeting 16S rRNA gene fragments

Three mesocosms were studied to evaluate the effect of wetland plants on the methanotrophic bacterial populations in the sediments of a full-scale constructed wetland. Cores were collected from two vegetated mesocosms and one unvegetated mesocosm from fall 2002 through summer 2003. Competitive quantitative PCR revealed no significant differences in the quantities of either Type I or Type II methanotrophic bacteria between the vegetated and unvegetated mesocosms. Type I methanotroph-biased nested PCR-DGGE resulted in the detection of 23 different populations related to Methylococcus, Methylomonas, Methylobacter, Methylocaldum, and Methylosarcina spp. Type II methanotroph-biased nested PCR-DGGE resulted in the detection of 5 different populations, more than 90% of which were related to previously uncultivated Type II methanotrophs. While wetland vegetation did not affect the structure of either the Type I or Type II methanotrophic communities, the Type I methanotrophic community structure was observed to vary seasonally. This work suggests that wetland plants neither enhanced nor adversely affected the size or structure of methanotrophic communities in our constructed wetland. Substantial quantities of both Type I and Type II methanotrophic populations were detected in both planted and unplanted mesocosms, suggesting that the constructed wetland had substantial potential for xenobiotic bioremediation whether or not plants were present.     

基于综合评价的矿业废弃地整治时序确定

为确定合理的矿业废弃地整治时序,保护生态环境,迎合矿产资源型城市经济发展和产业转型的用地需求。该文以北京市门头沟区为研究区域,从生态状况、修复成本、区位条件、经济和社会因素4个层面构建了确定矿业废弃地整治时序的评价指标体系,进而构建了基于该指标体系的评价模型。兼顾行政管理的需要,将门头沟区行政村界线图、煤矿废弃地分布图进行叠加,以村作为评价单元,对各指标进行量化,确定煤矿废弃地的整治时序。研究结果表明:在北京市门头沟区,煤矿废弃地分布在45个村庄,其中,近期整治的村为16个,煤矿废弃地面积121.94 hm2;中期整治的村为19个,煤矿废弃地面积105.41 hm2;远期整治的村为10个,煤矿废弃地面积54.27 hm2,该研究结果为该区矿业废弃地整治时序的确定提供了理论支持。     

Effects of ingested turmeric oleoresin on glucose and lipid metabolisms in obese diabetic mice: a DNA microarray study

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. Turmeric oleoresin, an extract of turmeric, is often used for flavoring and coloring. Curcuminoids and turmeric essential oil are both contained in turmeric oleoresin, and both of these fractions have hypoglycemic effects. In the present study, we comprehensively assessed the effect of turmeric oleoresin on hepatic gene expression in obese diabetic KK-Ay mice using DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR). Female KK-Ay mice aged 6 weeks (n = 6/group) were fed a high-fat diet containing turmeric oleoresin, curcuminoids, and essential oil for 5 weeks. The same diet without any of these fractions was used as a control diet. Ingestion of turmeric oleoresin and essential oil inhibited the development of increased blood glucose and abdominal fat mass, while curcuminoids only inhibited the increase in blood glucose. DNA microarray analysis indicated that turmeric oleoresin ingestion up-regulated the expression of genes related to glycolysis, beta-oxidation, and cholesterol metabolism in the liver of KK-Ay mice, while expression of gluconeogenesis-related genes was down-regulated. Real-time PCR analysis was conducted to assess the contribution of the curcuminoids and essential oil in turmeric oleoresin to the changes in expression of representative genes selected by DNA microarray analysis. This analysis suggested that curcuminoids regulated turmeric oleoresin ingestion-induced expression of glycolysis-related genes and also that curcuminoids and turmeric essential oil acted synergistically to regulate the peroxisomal beta-oxidation-related gene expression induced by turmeric oleoresin ingestion. These changes in gene expression were considered to be the mechanism by which the turmeric oleoresin affected the control of both blood glucose levels and abdominal adipose tissue masses. All of these results suggest that the use of whole turmeric oleoresin is more effective than the use of either curcuminoids or the essential oil alone.     

Mass spectrometry based sensor strategies for the authentication of oysters according to geographical origin

This study was undertaken to investigate the relevance of using the pyrolysis-MS (Py-MS) technique to discriminate the production area of oysters harvested over two years and to assess from the data of the second year of harvest the potential of an alternative MS-based technique, the solid phase microextraction-MS (SPME-MS), to perform this discrimination. Oysters were harvested in various areas of France, and models of discrimination according to harvest season were built from Py-MS fingerprints and from virtual SPME-MS fingerprints obtained by summing the mass spectra generated by the SPME-GC-MS system. The treatment of the Py-MS data by a 21-12-3 artificial neural networks led to a correct classification of only 89.2% of the oyster samples according to shoreline. The misclassifications thus did not allow use of the Py-MS technique as a relevant tool for authentication of oyster origin. The assessment of the potential of the virtual SPME-MS fingerprints to discriminate the production area of oysters was undertaken on a part of the sample set. The virtual SPME-MS data were pretreated according to two methods, filtering of raw data (FRD) and comprehensive combinatory standard correction (CCSC), a recently developed chemometric method used for the correction of instrumental signal drifts in MS systems. The results obtained with the virtual SPME-MS fingerprints are promising because this technique, when the data were pretreated by the CCSC method, led to a successful discrimination of the oyster samples not only according to shoreline but also according to production region. This study confirms that an efficient correction method (CCSC) of instrumental drifts can considerably increase the discriminative information contained in the volatile fraction of food products.     

Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

Consensus nematode 18S ribosomal DNA primers were designed by aligning available 18S sequences and identifying a variable region flanked by highly conserved regions. These primers were then used to amplify nematode 18S rDNA from whole soil community DNA extracted from a range of European grassland types. Cloning of the PCR amplicons (778 bp) followed by restriction digest analysis (RFLP) resulted in the recovery of 34 unique nematode sequences from the four grasslands studied. Comparison of these data with the limited number of 18S rDNA nematode sequences currently held in on-line databases revealed that all of the sequences could be assigned to known nematode taxa albeit tentatively in some cases. Two of the sequences recovered from the site in the Netherlands (wet, hay-grassland) were recovered in a clade that included a sequence of the genus Trichodorus whilst other sequences from this site showed similarity with 18S rDNA sequences of the genus Prismatolaimus (five sequences), Xiphinema (one sequence) and Enoplus (one sequence). Of the remaining sequences, two showed some affinity with Mylonchulus (UK, upland peat), four with Steinernema (UK) and one sequence with Mesorhabditis (Hungary, east European Steppe). Three sequences from the Netherlands and one from Hungary were recovered in a clade that included a sequence of the genus Pratylenchoides whilst three further sequences from the Netherlands and two from Hungary were recovered in a clade encompassing the genus Globodera. Of the remaining nine sequences, two (NL6, NL62) formed a distinct lineage within the Adenophorea with 90% bootstrap recovery in a paraphyletic clade that included sequences of Prismatolaimus and Trichodorus. Seven sequences (three from the Netherlands, three from the UK and one from Greece) were left unassigned though the tree topology suggested some relationship (58% bootstrap recovery) with the genus Cephalobus. To assess whether primers used to amplify 18S rDNA might be used to fingerprint genetic diversity in nematode communities in soil, the environmental sequence data were used to design a second set of primers carrying a GC-clamp. These primers amplified a 469 bp fragment internal to the region flanked by the primer set used to derive the nematode trees and were used to amplify 18S rDNA for subsequent analysis using denaturing gradient gel electrophoresis (DGGE). DGGE analysis of six major European grassland types revealed considerable genetic diversity between sites. However, the relationships seen with the DGGE data were inconsistent with previous studies where the same soils had been characterized with respect to functional and morphological diversity. To confirm that this second set of primers was amplifying nematode sequences, selected bands on the DGGE gels were extracted, PCR amplified and sequenced. The final alignment was 337 bases. These analyses revealed the presence of sequence signatures from the genera Paratrichodorus, Plectus, Steinernema, Globodera, Cephalobus and Pratylenchoides.     

Hepatic gene expression of the insulin signaling pathway is altered by administration of persimmon peel extract: a DNA microarray study using type 2 diabetic Goto-Kakizaki rats

Persimmon (Diospyros kaki) is a very popular fruit in East Asian countries, but its peels are not consumed despite the fact that they contain many antioxidants such as carotenoids and polyphenols. We prepared a fat-soluble extract from persimmon peel (PP) and fed type 2 diabetic Goto-Kakizaki (GK) rats an AIN-93G rodent diet supplemented with persimmon peel extract (PP diet) for 12 weeks. Compared with the control AIN-93G diet, the PP diet significantly reduced plasma glutamic-pyruvate transaminase activity, with accumulation of β-cryptoxanthin in the liver. DNA microarray analysis revealed that the PP diet altered hepatic gene expression profiles. In particular, expression of insulin signaling pathway-related genes was significantly enriched in differentially expressed gene sets. Moreover, Western blotting analysis showed an increase in insulin receptor beta tyrosine phosphorylation in rats fed the PP diet. These data suggest that the PP diet improves insulin resistance in GK rats.     

New method of DNA isolation from two food additives suitable for authentication in polymerase chain reaction assays

Locust bean gum and guar gum are galactomannans used as additives (E 410 and E 412, respectively) in the food industry as stabilizing agents. Analytical discrimination between the two additives in gums and foods is now feasible by molecular techniques. However, only complex and time-consuming DNA isolation protocols are available to date. We have developed simple improved protocols to obtain enough DNA suitable for PCR amplification from a few milligrams of commercial E 410 and E 412 additives (containing more than 75% polysaccharides). The suspension of additives in water or 10 mM Tris-HCl, pH 8.5, efficiently recovers DNA suitable for authentication in PCR assays. However, the Tris method was much more efficient for the extraction of DNA from E 410 than for E 412 additives. Conversely, the water method was the most suitable for detecting DNA extracted from E 412 or from E 410/E 412 mixtures. Combined with the use of the two specific ribosomal primer pairs previously designed, our methods are well-suited for a fast and simple high-throughput sample treatment of commercial gums for molecular certification.