Effects of drying methods on contents of bioactive compounds and antioxidant activities of Angelica dahurica.

Baizhi (Angelica dahurica) has been widely used as a traditional Chinese herbal medicine, functional food and cosmetic product ingredient, mostly because of the high furanocoumarin compounds in roots. Because the fresh root is perishable, drying techniques are needed to maintain a higher-quality product. Freeze-drying is the best method but energy-consuming and costly. The aim of this study was to analyze the quality (antioxidant and furanocoumarin content) of Baizhi roots after freeze-drying (the control) and in-the-shade, 40 and 70 °C drying. Antioxidant activity was revealed by 2,2-diphenyl-1-picrylhydrazyl and Fe2+ chelating assay, and the content of six furanocoumarin compounds, including xanthotoxin, bergapten, oxypeucedanin, imperatorin, phellopterin and isoimperatorin, was analyzed by liquid chromatography. Antioxidant activity was greater in roots with in-the-shade, 40 and 70 °C drying than freeze-drying. The furanocoumarin content pattern was similar with 70 °C drying and freeze-drying. A. dahurica roots dried at 70 °C may be an alternative method for maintaining high quality.

Influence of harvest stage on the pharmacological effect of Angelica dahurica.

BACKGROUND: Baizhi (Angelica dahurica) has been widely used as a traditional Chinese herbal medicine, functional food and cosmetic product ingredient, mostly because of the high furanocoumarin compounds in roots. The cropping system of Baizhi with its unique summer dormancy feature, is easily affected by the transition of its growth stages. The aim of this study was to analyze the quantity (size, form and dry weight [DW]) and quality (antioxidant and furanocoumarin content) of taproot and lateral root from three growth stages of Baizhi; vegetative (V-stage), summer dormancy (S-stage) and bolting stage (B-stage). RESULTS: Root length and diameter were lower at V-stage than the other two stages, and S-stage had higher lateral root to total root ratio. However, the highest root DW was observed at S-stage. Antioxidant activity was revealed by 2,2-diphenyl-L-picrylhydrazyl and Fe2+ chelating assay, and the content of six furanocoumarin compounds, including xanthotoxin, bergapten, oxypeucedanin, imperatorin, phellopterin and isoimperatorin, was analyzed by liquid chromatography. Although the antioxidant activity was less at S-stage than the other stages, furanocoumarin contents showed little variation. CONCLUSION: Considering the high DW and stable furanocoumarin composition, S-stage is the best harvest stage than the other stages because of its richer total pharmacological content.

The Ds1 Transposon Provides Messages That Yield Unique Profiles of Protein Isoforms and Acts Synergistically With Ds to Enrich Proteome Complexity via Exonization.

In exonization events, Ds1 may provide donor and/or acceptor sites for splicing after inserting into genes and be incorporated into new transcripts with new exon(s). In this study, the protein variants of Ds1 exonization yielding additional functional profile(s) were studied. Unlike Ds exonization, which creates new profiles mostly by incorporating flanking intron sequences with the Ds message, Ds1 exonization additionally creates new profiles through the presence or absence of Ds1 messages. The number of unique functional profiles harboring Ds1 messages is 1.3-fold more than that of functional profiles without Ds1 messages. The highly similar 11 protein isoforms at a single insertion site also contribute to proteome complexity enrichment by exclusively creating new profiles. Particularly, Ds1 exonization produces 459 unique profiles, of which 129 cannot be built by Ds. We thus conclude that Ds and Ds1 are independent but synergistic in their capacity to enrich proteome complexity through exonization.

Application of an inducible transposon with anther culture in generation of di-haploid homologous mutants.

BACKGROUND: Insertional mutagenesis represents one of the most effective ways to acquire information about a plant gene's function. However, it is hindered by the autosomal genome being diploid and therefore, most mutations being recessive. The problem is addressed by inducing the transposition during anther culture so that selected mutations can be transmitted and then regenerated to a homozygous state. RESULTS: To this end, we treated transgenic rice floral tissues containing the inducible transposon with an inducer, salicylic acid. Excision events were detected in regenerated calli and subsequent plantlets. DNA blot and PCR assay were used to determine the homogeneity of knockout mutants. About 5% of the mutants containing transposition events were homozygous. Furthermore, the inducible transposon was active during calli regeneration. CONCLUSIONS: This strategy could be applicable to improve transposition efficiency in microspore development stages to create stable di-haploid mutants in plants.

Analysis of new functional profiles of protein isoforms yielded by ds exonization in rice.

Insertion of transposable elements (TEs) into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization. Exonization can enrich the complexity of transcriptomes and proteomes. Previously, we performed a genome-wide computational analysis of Ds exonization events in the monocot Oryza sativa (rice). The insertion patterns of Ds increased the number of transcripts and subsequent protein isoforms, which were determined as interior and C-terminal variants. In this study, these variants were scanned with the PROSITE database in order to identify new functional profiles (domains) that were referred to their reference proteins. The new profiles of the variants were expected to be beneficial for a selective advantage and more than 70% variants achieved this. The new functional profiles could be contributed by an exon-intron junction, an intron alone, an intron-TE junction, or a TE alone. A Ds-inserted intron may yield 167 new profiles on average, while some cases can yield thousands of new profiles, of which C-terminal variants were in major. Additionally, more than 90% of the TE-inserted genes were found to gain novel functional profiles in each intron via exonization. Therefore, new functional profiles yielded by the exonization may occur in many local regions of the reference protein.

The extent of Ds1 transposon to enrich transcriptomes and proteomes by exonization.

BACKGROUND: Exonization is an event which an intronic transposed element (TE) provides splice sites and leads to alternatively spliced cassette exons. Without disrupting of the inserted gene's function, TEs can expand the proteome diversity by adding the splice variant that encodes a different, yet functional protein. Previously, we found that the main contribution of Ds exonization for gene divergence is not providing genetic messages but incorporating the intron sequences with different reading frame patterns to enrich the plant proteome. Ds1, another member of Ac/Ds transposon system, differs from Ds by providing 3 splice donor sites and 2 acceptor sites for alternative splicing, which may greatly increase the extent for proteome expansion. RESULTS: In this study, we performed a genome-wide survey of Ds1 exonization events to assess its extent to enrich proteomes in plants. Each Ds1 insertion yielded 11 transcript isoforms by integrating the splice donor and/or acceptor sites, which composed a bulk of all exonized transcript orthologs from the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice). The exonized transcripts were analyzed by the locations of the termination codon (PTC) and the putative targets for the nonsense-mediated decay (NMD) pathway were then excluded. Compared with the Ds element, Ds1 harbors more contents of non-NMD transcripts for protein isoforms. CONCLUSIONS: The contribution of Ds1 exonization for gene divergence is incorporating the intron sequences with different reading frame patterns to enrich the plant proteome. All these simulation results direct new experimental analysis at the molecular level.

Genome-wide survey of ds exonization to enrich transcriptomes and proteomes in plants.

Insertion of transposable elements (TEs) into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization which can enrich the complexity of transcriptomes and proteomes. Previously, we performed the first experimental assessment of TE exonization by inserting a Ds element into each intron of the rice epsps gene. Exonization of Ds in plants was biased toward providing splice donor sites from the beginning of the inserted Ds sequence. Additionally, Ds inserted in the reverse direction resulted in a continuous splice donor consensus region by offering 4 donor sites in the same intron. The current study involved genome-wide computational analysis of Ds exonization events in the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice). Up to 71% of the exonized transcripts were putative targets for the nonsense-mediated decay (NMD) pathway. The insertion patterns of Ds and the polymorphic splice donor sites increased the transcripts and subsequent protein isoforms. Protein isoforms contain protein sequence due to unspliced intron-TE region and/or a shift of the reading frame. The number of interior protein isoforms would be twice that of C-terminal isoforms, on average. TE exonization provides a promising way for functional expansion of the plant proteome.

Ds transposon is biased towards providing splice donor sites for exonization in transgenic tobacco.

Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization, which can enrich the complexity of transcriptomes and proteomes. In this study, the first exonization event was detected when the modified rice EPSPS marker gene was inserted with the Ac transposon 5' end, which provided a splice donor site to yield abundant novel transcripts. To assess the contribution of splice donor and acceptor sites of transposon sequences, we inserted a Ds element into each intron of the EPSPS marker gene. This process yielded 14 constructs, with the Ds transposon inserted in the forward and reverse direction in each of the 7 introns of the EPSPS marker gene. The constructs were transformed into tobacco plants, and novel transcripts were identified by RT-PCR with specific primers. Exonization of Ds in EPSPS was biased towards providing splice donor sites of the inserted Ds sequence. Additionally, when the Ds inserted in reverse direction, a continuous splice donor consensus region was determined by offering 4 donor sites in the same intron. Information on these exonization events may help enhance gene divergence and functional genomic studies.

A one-time inducible transposon to create knockout mutants in rice.

Use of a transposon is an efficient tagging tool for exploring the function of the gene it inserts into or is adjacent to. A few modifications have been applied to the native Ac transposon to allow it to transpose efficiently or spontaneously and stop quickly thereafter. Furthermore, locating the transposon between a constitutive plant promoter and a reporter gene, such as the firefly luciferase gene, allows for nondestructively detecting excision events in vivo. This chapter describes a detailed protocol for one-time inducible transposon tagging of rice cells and their subsequent screening and regeneration into mutant lines.

A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase.

The piggyBac (PB) transposable element has recently accumulated enormous attention as a tool for the transgenesis in various eukaryotic organisms. Arginine-rich cell-penetrating peptides (CPPs) are protein transduction domains containing a large amount of basic amino acids that were found to be capable of delivering biologically active macromolecules into living cells. In this study, we demonstrate a strategy, which we called "transposoduction", which is a one-plasmid gene delivery system mediated by the nontoxic CPP-piggyBac transposase (CPP-PBase) fusion protein to accomplish both protein transduction and transposition. CPPs were proven to be able to synchronously deliver covalently linked PBase and noncovalently linked a cis plasmid into human cells. The expression of promoterless reporter genes coding for red (dTomato) and yellow (mOrange) fluorescent proteins (RFP and YFP) with PB elements could be detected in cells treated with the PBase-expressing plasmid after 3 days indicating transposition of coding regions to downstream of endogenous promoter sequences. An enhanced green fluorescent protein (EGFP) plasmid-based excision assay further confirmed the efficiency of the bifunctional CPP-PBase fusion protein. In conclusion, this strategy representing a combinational concept of both protein transduction and mobile transposition may provide tremendous potential for safe and efficient cell line transformation, gene therapy and functional genomics.

Inhibition of allergen-induced airway inflammation and hyperreactivity by recombinant lactic-acid bacteria.

Recombinant lactic-acid bacteria (LAB) are able to inhibit allergen-specific T-cell responses. In this study, we examined whether oral feeding of recombinant LAB was able to suppress allergen-induced airway inflammation and hyperreactivity (AHR) in a murine model. Animals were intraperitoneally sensitized with Dermatophagoides pteronyssinus group-5 allergen (Der p 5) and orally treated with recombinant LAB containing a plasmid-encoded Der p 5 gene or placebo on day 7 and day 14 for three days consecutively. Twenty-one days after sensitization, mice underwent inhalational challenging. Der p 5-specific immunological responses including changes to specific immunoglobulin G and E (IgE) levels, the presence of cells in the bronchoalveolar lavage fluid (BALF), and AHR were assessed following this inhalational challenge. We demonstrated that oral feeding of recombinant LAB could significantly decrease the synthesis of Der p 5-specific IgE, and AHR. Furthermore, following such treatment, we also noted that both neutrophils and eosinophils had infiltrated the BALF to a significantly lower extent, when compared to the vehicle-treated group. Neither recombinant allergen nor LAB alone was able to suppress allergen-induced immune responses. Our findings suggest that treatment with recombinant LAB at a low dose can suppress allergen-induced airway allergic inflammation, this providing a basis for developing a novel therapeutic method for allergic airway diseases.