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BackgroundObesity is a common health problem among patients with schizophrenia, but the precise mechanisms are not fully understood. There has been much interest in the relationship between gut microbiome and development of obesity. Gender-dependent microbial alteration has been reported in previous studies. However, the gender factor in gut microbiome composition of schizophrenia patients has been less investigated. Our study aimed to identify differences in gut microbiota between schizophrenia patients with normal weight and central obesity and investigate the gender specific features.MethodTwenty participants (10 males, 10 females) with central obesity (CO) and 20 participants (10 males, 10 females) with normal weight (NW) were recruited from two rehabilitation wards in a psychiatric hospital in central Taiwan. Fecal samples from 40 participants were processed for microbiota analysis. The intestinal microbiota composition was analyzed using next-generation sequencing and QIIME software.ResultsSignificantly higher richness of gut microbiota at the class level (measured by the number of observed OTUs) was observed in female NW subjects than in female CO subjects (P = 0.033). Furthermore, female NW subjects showed higher alpha diversity at both phylum and class levels (measured by the Shannon, Simpson, and Inverse-Simpson indexes) compared with female CO subjects. Males showed no significant difference in alpha diversity between groups. Taxonomic analysis showed that female CO subjects had significantly lower abundance of Verrucomicrobia (P = 0.004) at the phylum level, reduced abundance of Akkermansia (P = 0.003) and elevated level of Prevotella (P = 0.038) and Roseburia (P = 0.005) at the genus level.ConclusionsThe present results evidenced altered microbiome composition in schizophrenia patients with central obesity and further suggested the role of the gender factor in the process of gut dysbiosis.

This paper investigated the DNA barcoding of indigenous cinnamon. The essential oil composition of leaves and the partial non-coding internal transcribed spacer (ITS2) region of ribosomal genes from indigenous cinnamon plants (Cinnamomum osmophloeum Kaneh.) were investigated. Leaf essential oils from seven geographical strains of C. osmophloeum Kaneh. were obtained by hydrodistillation and characterized by gas chromatography-mass spectrometry. Six chemotypes, including cinnamaldehyde, cinnamaldehyde/cinnamyl acetate, cinnamyl acetate, linalool, camphor and mixed, were identified. Seven representative geographical strains were further used to study their genetic diversity based on the partial ITS2 region using PCR amplification and DNA sequencing to evaluate the region's use in the application of DNA barcoding C. osmophloeum Kaneh. A phylogenetic tree was constructed using UPGMA clustering method. Our results indicate that the partial ITS2 nucleotide sequences for all seven geographical strains are identical and not correlated with essential oil composition. The partial ITS2 sequences is sufficient for barcoding C. osmophloeum Kaneh., while that additional genes will need to be analyzed to identify samples of various chemotypes. We believe that this partial ITS2 locus serves as a good starting point for large-scale testing of DNA barcoding of other plant species across Taiwan.

Estimation of the genome size of Phalaenopsis species is important because this orchid is employed in various research areas including variety improvement and studies of molecular genetics. In this research, the ovary tissue of the orchid flower before pollination was used to estimate the nuclear DNA content (2 C value) by flow cytometry for 50 species of four subgenera with eight sections of the genus Phalaenopsis (2 n = 2 x = 38). Measurements were taken by repeated sampling of the fresh flowers collected from several germplasm banks in Taiwan. The 2 C values of 50 species ranged from 2.77 pg in P. philippinensis to 17.47 pg in P. lobbii and varied at different degrees within each subgenus/section. The nuclear DNA contents of species in sections Phalaenopsis and Fuscatae were homogenous without significant difference while those in sections Zebrinae and Amboinenses were highly variable. Subgenera Parishianae and Aphyllae and two sections ( Esmeralda and Deliciosae ) of subgenus Phalaenopsis showed higher mean 2 C values than those of other sections. Intraspecific variations in 2 C values were observed in some species. The information provided by the present research on genome size of Phalaenopsis species together with their geographic distribution and studies on molecular phylogeny would give further insight to plant systematics of this genus. At the same time, it would be helpful to orchid breeders to increase the efficiency of their improvement programs.

The bi-functional nuclease AtBFN2 from Arabidopsis thaliana (EC 3.1.30.1) depends on zinc ion for cleaving single stranded DNA and RNA to yield 5′-nucleotides. It is a glycoprotein that participates in plant development and differentiation. The crystal structure of AtBFN2 shows a bound sulfate ion in the active site, at the center of the tri-nuclear cluster of zinc ions. The protein folds into a mostly α-helical structure with five short β-strands and contains four disulfide bonds. The zinc ions are coordinated to the side chains of three Asp and five His residues, two backbone atoms of Trp1, the sulfate ion, and a water molecule. An adenine base is bound adjacent to the active site and stacks with Tyr59. The core sugar residues attached to the three N-glycosylation sites of Asn91, Asn110 and Asn184 are also observed. By comparison with the nuclease P1 structure (PDB ID: 1AK0), the AtBFN2-sulfate-adenine complex model suggests a similar catalytic mechanism, in which the reaction starts with in-line attack at the phosphate by a zinc-activated water molecule.

Alternative medicinal agents, such as the herb Bupleurum, are increasingly used in modern medicine to supplement synthetic drugs. First, we present a review of the currently known effects of triterpene saponins-saikosaponins of Bupleurum species. The putative biosynthetic pathway of saikosaponins in Bupleurum species is summarized, followed by discussions on identification and characterization of genes involved in the biosynthesis of saikosaponins. The purpose is to provide a brief review of gene extraction, functional characterization of isolated genes and assessment of expression patterns of genes encoding enzymes in the process of saikosaponin production in Bupleurum species, mainly B. kaoi. We focus on the effects of MeJA on saikosaponin production, transcription patterns of genes involved in biosynthesis and on functional depiction.

Additional file 1: Figure S1. The AVRDC breeding program of mungbean.

The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.

A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to ∼30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

Endopolyploidy is frequently observed during development in plant species. Patterns of endopolyploidy are diverse in the various organs of different plant species. However, little is known about the role of endopolyploidization and its significance in orchids. This study was undertaken to determine the extent of endopolyploidy in different tissues of the diploid and tetraploid genotypes of Phalaenopsis aphrodite subsp. formosana and to examine the factors that contribute to increased ploidy levels. Endopolyploidy occurs in various tissues of diploid and tetraploid orchids, at different developmental stages and under different culture conditions, as determined by flow cytometry. In this study, different patterns of endopolyploidy were observed in parts of the protocorms, leaves, roots and flowers. Endopolyploidy was found in all tissues studied except the pollinia and the tetraploid ovaries. A higher degree of endopolyploidy was observed in mature tissues compared to young tissues, greenhouse-grown plants compared to in vitro plants and diploid plants compared to tetraploid plants. We discuss the relationships between endopolyploidization and several factors related to plant growth, as well as some practical considerations of these findings.

Three novel ethylene response factor (ERF) genes, BkERF1, BkERF2.1 and BkERF2.2, were isolated from a medicinal plant, Bupleurum kaoi. The deduced BkERFs contain a canonical nuclear localization signal and an ERF/AP2 DNA binding domain. RNA gel blot analysis revealed that BkERF1 and BkERF2.1 were ubiquitously expressed at low levels in all parts of mature plants, and that BkERF2.2 was expressed at moderate levels in vegetative tissues. Exogenous application of methyl jasmonate induced BkERF1/2.1/2.2 transcripts. BkERF2.2 transcript levels were slightly increased by addition of ethephon and salicylic acid. BkERFs were localized in the plant nucleus and functioned as transcriptional activators. In B. kaoi cells overexpressing BKERFs, inoculation with Botrytis cinerea increased expression of some defense genes which are associated with enhanced disease resistance. Similarly, overexpression of BkERFs in transgenic Arabidopsis thaliana resulted in elevated mRNA levels of the defense gene PDF1.2, and in enhanced resistance to B. cinerea. Collectively, these results provide evidence that BkERFs mediate the expression of defense-related genes in plants.

The conversion of flavonoid aglycones to their glycosides by plant glycosyltransferases may affect a wide range of outcomes, including stability, solubility and bioavailability. Scutellaria barbata, rich in flavonoid glycosides, is widely used as a traditional Chinese herbal medicine. In this study, a flavonoid glycosyltransferase cDNA (SbUGT) and its promoter from S. barbata were cloned and characterized as a flavonoid glycosyltransferase using whole-cell biotransformation. Fragments of different lengths of the 5'-flanking region of the SbUGT gene were fused to the beta-glucuronidase (GUS) gene and analyzed with transgenic Arabidopsis plants using histochemical and fluorometric assays. GUS activity in transgenic plants carrying the SbP-850U construct (-850 to +86 relative to the transcription start site) displayed the highest level and was enhanced by salt and methyl jasmonate, similar to the expression patterns of the endogenous SbUGT. GUS activity disappeared when the promoter was deleted to -98, and deletion analyses indicated the existence of positive and negative regulatory element(s). Unexpectedly, plants carrying the construct SbP-102U (-102 to +86) exhibited strong GUS activity exclusively in the roots. Our experiments revealed that the specific expression is mediated by different promoter regions and the unique region driving root-preferred expression can be used as a root-specific promoter.

To establish a mass micropropagation procedure for Cephalotus follicularis, the effects of varying the strengths of solid Murashige and Skoog (MS) medium were investigated using subcultured shoot explants. After a 60-day primary culture from root mass, the regenerated shoot explants were subcultured every 60 days in solid MS medium. To facilitate shoot proliferation, liquid MS medium was applied with or without exogenous auxin and cytokinin. Our results demonstrate that shoot proliferation and survival of C. follicularis is most effective in modified MS (MMS) medium containing one-fifth or one-tenth strength macronutrients and full-strength micronutrients. Successful shoot proliferation and development of C. follicularis explants were obtained in one-fifth or one-tenth modified liquid MS medium without auxin and cytokinin or with addition of 5 μM indole 3-acetic acid/1 μM N6-benzyladenine for 45 days. The liquid medium consistently produced more explants than the solid medium and shortened the culturing time. Plantlets cultured in hormone-free one-fifth MMS medium developed greater root systems. Using the liquid culture we established, vigorous plants with extensive roots were obtained within 4 months. Plant survival in the greenhouse reached 100%.

Standardization and modernization of Chinese medicinal herbs are limited partially by misidentification of processed materials. Our goal was to develop an efficient method for verification of Chinese medicinal herbs, based on the variable sites of the rDNA internal transcribed spacer (ITS) region. We analyzed sequence differences in ITS of three Bupleurum species, B. kaoi Liu Chao et Chuang, B. falcatum L. and B. chinense DC., and developed a rapid detection method using a sequence-specific oligonucleotide probe (SSOP) array. The SSOP array, composed of poly-T tailed sequence-specific oligonucleotides, was hybridized to the digoxigenin (DIG)-labeled target ITS DNA of the Bupleurum species. The detected signals corresponded precisely to the specific sequences. This array provides a reliable and economical method for authenticating a large number of Chinese medicinal herbs. The short duration of the procedure (within 30 h) makes it an especially useful tool in verifying processed plant material.

Although mungbean (Vigna radiata (L.) Wilczek) is commonly used as human food; the genomic resources of this species available in databases are limited. This study aims to develop expressed sequence tag (EST) resources for mungbean genes informative to early seedling development and chilling response. Two mungbean varieties that differ in disease resistance were found to also differ in their susceptibility to chilling temperatures. A total of 1,198 ESTs were obtained from one cDNA library and four PCR-select cDNA subtraction libraries; among these 523 were clustered into 136 contigs and 675 were singletons. The 811 non-redundant uniESTs were compared to GenBank using the Basic Local Alignment Search Tool (BLAST) and WU-BLAST algorithms, of these only 489 uniESTs had significant sequence homology, which may be involved in resuming the metabolic activity of seedlings, switching on photomorphogenesis, fuelling photosynthesis and/or initiating the unique developmental programs. Their encoded proteins may associate with regulatory proteins to trigger a direct stress response or participate in acclimation to environmental stressors. The uniEST platform reported will enrich the genomic resources of mungbean for functional genomic research on seedling development and chilling response of tropical crops and provide targets for improving the chilling tolerance of the tropical crops.

Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.

The plant regulator methyl-jasmonate (MeJA) mediates diverse developmental processes and defense responses which induce a variety of secondary metabolites. This study reports the regulatory network of MeJA-induced genes in adventitious root cultures of the medicinal herb Bupleurum kaoi. A cDNA microarray containing 465 unique expressed sequence tags (ESTs) as probe sets, generated with a PCR-Select cDNA Subtraction Kit, was employed to profile the transcriptome of MeJA induction at thirteen points in time. Real-time PCR was performed to verify changes in expression of 36 unique ESTs (uniESTs), detected via microarray. Genes upregulated by MeJA include those involved in saikosaponin, amino acid, phenylpropanoid and jasmonate biosynthesis, defense signaling, plant hormone regulation and stress protection; indicating that the JA signaling pathway interacts with other signaling pathways leading to transcriptional reprogramming in B. kaoi adventitious roots. Some of these MeJA-induced genes have applications in agricultural, pharmaceutical and food industries.

Endoreduplication, the replication of nuclear DNA without subsequent mitosis and cell division is an important component of organ development and is often related to plant cell size. Little is known, however, about the effect of sub-optimal temperatures on the endoreduplication and floral development. We analyzed ploidy levels, cell weight and cell number during flower development in Phalaenopsis aphrodite and Oncidium varicosum grown at 25 and 15 °C. Low temperature decreased the growth rate and the endoreduplication transition rate. A longer growth period compensated for the decreased growth rate so that the distribution of ploidy levels similar at both growth temperatures. Low temperature decreased growth rates and endoreduplication transition rates for both orchid species. These results are consistent with previous studies that endoreduplication cycle may use the same molecular machinery for their DNA replication as that used during the S-phase of the mitotic cell cycle. In both species, the average C value is positively correlated with the average cell fresh weight. However, 15 °C increased the ratio of average C value to average cell fresh weight in P. aphrodite but decreased the ratio in O. varicosum. Although ploidy level plays a role in final cell size, growth temperature modulates the effect ploidy level on final cell weight.

The role of small heat shock proteins (sHsps) in the cellular chaperone system was examined with application of the feedback control technique widely used in engineering for stabilizing a specific system. We proposed a dynamic feedback control model for the thermotolerance machinery of sHsps from the system biology perspective, and for the first time verified that a system stabilization feedback control scheme is involved in the chaperone protecting activity under heat shock conditions. In the dynamic model of the chaperone protection system, the defense mechanism of sHsps is a sigmoid function and the influence of environment temperatures is a bell-shaped function. The close fit of the system dynamic behaviors to the kinetic data suggests that our system stabilization control model matches the protective mechanism conveyed by sHsps. Furthermore, the temporal changes of the unfolded intermediates and the sHsp-substrate complexes can be predicted by the proposed dynamic stabilization model, which are not easily measured in biochemical experiments. The identified feedback control matrix can be viewed as a biochemical interaction for measuring the protective activity of sHsps qualitatively and quantitatively. Our work introduces an innovative approach for analysis and detecting the kinetics of sHsps to resist protein aggregation from the system stabilization point of view.

Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However, a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate crystals.

We isolated three mungbean Hsc70 cDNAs (VrHsc70-1, 70-2 and 70-3) and characterized their developmental expression at both the transcript and protein levels. We also characterized the binding specificity between each VrHsc70 protein and its potential substrates. RNase protection assays showed that these three cytosolic VrHsc70 genes were expressed similarly in all organs at all times during the mungbean life cycle, except at the initiation of germination and during late seed embryogenesis. Western blotting analyses showed that a different group of cytosolic VrHsc70 proteins accumulated in dehydrated seeds during seed maturation and the accumulated proteins remained high during the early stages of germination. Binding specificities of these three mungbean Hsc70s were determined using the C-terminal 30 kDa of the three VrHsc70s to select bound heptapeptides using phage display screening, and were confirmed by ELISA. We found that the heptapeptides, KVWVLPI, KLWVIPQ and YAPLSRL, specifically bound to the C-terminal 30 kDa region of VrHsc70-1, 70-2 and 70-3, respectively. The hydrophobic residues in the core of the heptapeptides, as well as residues 6 and 7, might contribute to the binding specificity. Our results indicate that the function of these three VrHsc70s may not be important in seed maturation or in desiccation tolerance, but are more likely involved in normal growth and development.

Endoreduplication, a process to amplify nuclear DNA without cell division, is widespread in plants. This study aims to formulate endoreduplication process as a dynamic system using orchid flowers in favor of their slow growth rate. We analyzed the ploidy levels during orchid flower development and proposed an improved model to describe the relationship between endoreduplication and cell growth. Our model combined a logistic growth model with an endoreduplication model of Schweizer et al. [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7070]. We found that using the Fermi function to describe the transition rates from one C value to next higher C value significantly improved simulation of changes in growth and endoreduplication. The growth rate, endoreduplication transition rates, and nuclei number of each C level at different developmental stages were computed. Our results indicated that cells with higher C values had lower transition rates and less potential for further endoreduplication, and the time that endoreduplication stopped occurred at the same time flower fresh weight stopped increasing. In addition, average cell fresh weight was positively correlated to average C value, suggesting that endoreduplication is a contributing factor to cell growth.

Karyotypes of nine Phalaenopsis species and the closely related Doritis pulcherrima were compared based on Feulgen- and DAPI-stained somatic metaphase chromosomes prepared from root tips. All species had the same chromosome number (2n\equals 2x\equals 38) , but their karyotypes differed markedly in absolute chromosome size, relative chromosome size, and the position of the centromere. Both genome size and the amount of constitutive heterochromatin (CH) varied widely among the species studied, and there was a positive correlation between these two parameters. The distribution of CH in the genomes was non-random: one or both arms of long chromosomes usually possessed large blocks of CH, while the small chromosomes contained little or no CH. DAPI-staining revealed that most CH regions are rich in AT base pairs. We suggest that differential accumulation of CH is a major cause for karyotype variation in Phalaenopsis orchids.

Nuclear DNA contents were estimated by flow cytometry in 18 Phalaenopsis Blume species and Doritis pulcherrima Lindl. DNA amounts differed 6.07-fold, from 2.74 pg/diploid nuclear DNA content (2C) in P. sanderiana Rchb.f. to 16.61 pg/2C in P. parishii Rchb.f. Nuclear DNA contents of P. aphrodite Rchb.f. clones, W01-38 (2n = 2x = 38), W01-41 (2n = 3x = 57), and W01-22 (2n = 4x = 76), displayed a linear relationship with their chromosome numbers, indicating the accuracy of flow cytometry. Our results also suggest that the 2C-values of the Phalaenopsis sp. correlate with their chromosome sizes. The comparative analyses of DNA contents may provide information to molecular geneticists and systematists for genome analysis in Phalaenopsis. Endoreduplication was found in various tissues of P. equestris at different levels. The highest degree of endoreduplication in P. equestris was detected in leaves.

The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1) encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+)-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC)/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+)-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

To apply random amplified polymorphic DNA for analysis of phylogenetic relationships, we used 34 synthetic oligonucleotides as primers to examine interspecific and intraspecific variations among 18 genotypes, nine species ofNicotiana. The nine species used in this study belong to sectionsTomentosae andAlatae. In addition, we attempted to clarify the taxonomic position ofN. sylvestris. A total of 354 distinct DNA fragments were obtained by polymerase chain reaction. Pair-wise comparisons of unique and shared amplification products were used to generate Jaccard's similarity coefficients and Nei and Li's similarity coefficients with the computer software of numerical taxonomy and multivariate analysis system. On the basis of the dendrogram constructed with the similarity coefficients, the 18Nicotiana genotypes were divided into two clusters. The classification analyzed by RAPD markers is in accordance with the classification of Goodspeed thatN. sylvestris is a member of sectionAlatae.

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes.

Field observations suggest that Phaseolus acutifolius A. Gray is more heat and drought tolerant than P. vulgaris L. Since high temperature stress and water stress are difficult to separate in field experiments, we do not know if P. acutifolius is more high temperature tolerant than P. vulgaris. The goal of our research was to determine whether P. acutifolius is more heat tolerant than P. vulgaris in the absence of water stress. Plants grown at 25°C for 13 d in controlled environment chambers were separated into two groups ; one group was maintained at 25°C for 8 d and the other at 32°C for 8 d. Air vapor pressure deficit at the two temperatures was maintained at 1.1 kPa. Phaseolus vulgaris growth was significantly decreased at 32°C compared with growth at 25°C. The decrease included total leaf area, total dry weight, root weight, and net assimilation rate. Phaseolus acutifolius grew similarly at both temperatures. Separate experiments investigated the effects of temperature on seed germination and early seedling growth. Seed germination of both species was reduced by about 11% at 32°C compared with germination at 25°C. Initial P. vulgaris radical growth was inhibited at 32°C whereas radical growth of P. acutifolius was not affected. Our results indicate that at the tissue level P. aculifolius is more heat tolerant than P. vulgaris.

The biosystem includes microbe, suspended cells of plants, transgenic organs, and other transforming systems. The biological transformation is to use the enzymes produced by the biosystem for catalyzed reactions of exogenous compounds, which has been widely used in the structural modifications of secondary metabolic products of plants, the search for the active leading compounds, and the explorations of drug structure-activity relations. This is called green chemistry.

In a previous paper we reported the isolation of streptomycin-resistant mutants from Nicotiana plumbaginifolia and presented evidence for chloroplast control of the resistance trait. To understand the molecular basis of the resistance in these mutants, we sequenced three regions in the chloroplast 16s rRNA gene, which correspond to the 5' terminus, the 530 loop, and the 900 stem/loop of Escherichia coli 16s rRNA, and compared them with the sequences of the wild-type. Our results show that: (1) nine mutants have a C to T change at position 912, (2) one mutant (SR1021) has a G to A change at position 885, (3) one mutant has a C to T change at position 526, based on E. coli numbering; and (4) three mutants do not have any change in the regions analyzed. The point mutation detected in SR1021 has not been reported previously. In E. coli 16s rRNA, position 885 is protected from chemical probing by ribosomal protein S12 and is closely juxtaposed with the streptomycin-binding region (positions 912-915) in the predicted secondary structure. It is likely that the G to A transition at this position is a novel mutation for streptomycin resistance.

FK228 (depsipeptide) is a natural prodrug that inhibits class I histone deacetylases. We aimed to investigate the effects FK228 has on prostate cancer cells in vivo. In non-obese diabetic-severe combined immunodeficient mice implanted with human prostate cancer cells, 50 mg/kg FK228 given orally 3 times a week inhibited tumor growth and metastasis. The median time to the experimental end point (tumor volume 2 cm3 or death) in the untreated group was 52 days, and average tumor volume was 0.8 ± 0.18 cm3. At the same time, 94.4% of FK228-treated mice survived and had average tumor volumes of 0.37 ± 0.1 cm3. All untreated animals died at 98 days, whereas, 61% of treated animal remained alive. Sizeable metastatic tumors positively stained for prostate-specific antigen (PSA), and limited air gaps were found in the lungs of untreated mice. In animals treated with FK228, lung morphology appeared normal. Primary tumors of treated animals were highly positive for PSA, and had an increased level of p21 and the proapoptotic protein Bax. Sections taken from FK228-treated animals and examined under an electron microscope showed condensed chromatin and apoptotic bodies. PSA serum levels were higher in untreated than in treated animals and correlated with tumor volume. Because prolonged oral administration of 50 mg/kg or a single oral dose of 1.2 g/kg FK228 did not cause adverse effects and inhibited proliferation of human prostate cancer cells in vivo, FK228 likely has a potential anticancer effect for prostate cancer.

Two full-length lipid transfer protein (LTP) cDNAs were isolated from mungbean (Vigna radiata) and designated Vrltp1 and Vrltp2. The deduced amino acid sequences contain the two highly conserved pentapeptides characteristic of plant LTPIs suggesting these Vrltps belong to the LTPI gene family. Vrltp1 mRNA was detected in developing seeds, but Vrltp2 mRNA was not. Within the vegetative tissues, the Vrltp1 and Vrltp2 mRNAs were present only in leaves and stems, but not root tips. Salt and dehydration stresses and exogenous abscisic acid (ABA) treatments resulted in increased mRNA levels of both Vrltps in leaves. We suggest that these unique Vrltps are specific to growing shoot tissues, and may play an important role in plant acclimation to water stress.

Lactoferrin, a ferric binding glycoprotein found in milk, can possibly prevent microbial infection of the mammary gland and gastrointestinal tract. To define the regulation of the porcine lactoferrin gene ( pLTF ), we cloned its 5′-flanking region from a porcine liver genomic library and analyzed the 5′ upstream region of approx. 4 kb, two exons, and an intron. The transcription start site was localized by primer extension to residue G, which is 41 nucleotides upstream from the ATG start codon. The pLTF 5′-flanking region possesses several putative cis-acting regulatory elements found in both housekeeping and inducible genes; to define their function, they were inserted into a chloramphenicol acetyltransferase reporter construct. The region up to −156 sufficed for basic promoter activity, whereas the region up to −780 was required for maximal promoter activity in porcine testis cells (ST cells), kidney cells (PK15 cells) and human mammary epithelial cells (HBL-100 cells). Detailed analysis of this proximal region by DNase I footprinting and electrophoretic mobility shift assays reveals that the ubiquitous factors SP1, AP2 and the mammary gland-specific factor (MGF) might play significant roles in regulating the transcription of the pLTF gene.

A tomato hsc70 genomic clone (Lehsc70-3; Lycopersicon esculentum heat-shock cognate 70-3) was obtained by screening a genomic library with the tomato Lehsc70-2 cDNA. Two restriction fragments of 2.6 and 5 kb, which compose the Lehsc70-3 gene, were subcloned into pBluescriptIIKS+ and analyzed. Transcript mapping reveals that the mature Lehsc70-3 mRNA contains a 122-nt 5' untranslated region (UTR), a coding region of 1956 nt corresponding to a polypeptide of 651 amino acids, an intron of 717 nt and a 3′ UTR. Analysis of genomic DNA indicates that Lehsc70-3 is present as a low-copy-number gene. We note that the sequence upstream from the coding region of Lehsc70-3 shares common features with a number of hsc/hsp genes. High-temperature treatment (37°C) caused a twofold increase in the level of the Lehsc70-3 mRNA. However, Lehsc70-3 has also been expressed at substantial levels in tomato vegetative tissues, suggesting a general function of this hsc70 gene in L. esculentum

In an effort to understand the mechanism of streptomycin resistance in Nicotiana plumbaginifolia, we have sequenced the chloroplast rps12 gene, a potential molecular target. We report that a streptomycin-resistant mutant isolated from protoplast cultures of N. plumbaginifolia contains an A-to-G transition at nucleotide position 149 in exon 2 of the chloroplast rps12 gene. The detected point mutation predicts a substitution of arginine for lysine in a phylogenetically conserved region.

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25 degrees C and 32 degrees C. Mitochondria isolated from P. vulgaris plants grown at 32 degrees C had reduced electron transport and were substantially uncoupled. Growth at 32 degrees C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25 degrees C grown plants. Mitochondria isolated from 25 degrees C grown P. vulgaris plants measured at 42 degrees C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42 degrees C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.

The relationship between abscisic acid (ABA) and leaf water status was studied during the air drying of detached leaves of eastern cottonwood (Populus deltoides Marsh.). The ABA content increased exponentially as leaf water potential and leaf turgor potential decreased. No clearly defined thresholds were observed between ABA content and these variables. ABA content was linearly related to the relative fresh weight and was not related to the osmotic potential.