Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

FISH and GISH analysis of the genomic relationships amongPanax species

Ginseng is a well-known medicinal plant that has been used as an anti-aging agent for many years in East Asia. In the genusPanax, only three species,P. ginseng (Oriental ginseng),P. quinquefolius (American ginseng) andP. notoginseng (Chinese ginseng), are currently considered to be important medicinal herbs. Despite the increase in their breeding value, molecular cytogenetic information on the species is not available. To analyze the genomic relationships among thePanax species, FISH (fluorescencein situ hybridization) and GISH (genomicin situ hybridization) techniques were applied. FISH analysis revealed that the 45S and 5S rRNA genes ofP. notoginseng (2n=2x=24) andP. ginseng (2n=4x=48) cluster on a single locus on different chromosomes, whileP. quinquefolius (2n=4x=48),P. japonicus (2n=4x=48), and Korean wild ginseng (2n =4x= 48) had one locus of the 45S rRNA gene and two loci of the 5S rRNA gene, respectively. GISH analysis using genomic DNA as a probe detected strong cross-hybridization of genomes betweenP. ginseng andP. quinquefolius. GISH analysis of other species showed weak or no distinct signals on the chromosomes. Our data revealed thatP. ginseng andP. quinquefolius showed the highest degree of homology, indicating that these species diverged in most recent years.     

Application of an airlift bioreactor system for the production of adventitious root biomass and caffeic acid derivatives of Echinacea purpurea

In this study, we evaluated the feasibility of using mass cultivation of the adventitious roots of Echinacea purpurea in balloon type bubble (air-lift) bioreactors to produce caffeic acid derivatives, which have pharmaceutical and therapeutic values. An approximately 10 fold increase in biomass and secondary compounds was observed after 4 weeks of culture in balloon type bubble bioreactors (5 L capacity containing 4 L of half strength MS medium). In addition, a linear relationship was observed between the concentration of biomass and the sucrose and ion consumption rate. Furthermore, the concentration of biomass in the bioreactor culture was found to increase as the conductivity decreased. An inoculum density of 7 g/L FW and an aeration rate of 0.1 vvm were found to be suitable for inducing the accumulation of biomass and secondary metabolites. Of the three caffeic acid derivatives evaluated (caftaric acid, chlorogenic acid, and cichoric acid), the concentration of cichoric acid was the highest (26.64 mg/g DW).     

Linoleic and α-linolenic fatty acids affect biomass and secondary metabolite production and nutritive properties of Panax ginseng adventitious roots cultured in bioreactors

The effect of elicitation with linoleic (C18:2) and α-linolenic (C18:3) fatty acids (LLA and α-LNA) was investigated in Panax ginseng C.A. Meyer adventitious roots cultured in 5 l balloon-type bioreactors. Fatty acids were added in culture medium at 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μmol l⁻¹ at day 40, at the end of exponential growth phase. Roots were harvested and assayed at day 47. Elicitation with both LLA and α-LNA enhanced accumulation of total polyphenolics and flavonoids in roots compared with control without elicitation. The highest accumulation of flavonoids was observed at 5.0 μmol l⁻¹ for both elicitors. Total phenolics production reached its highest value of about 4.0 mg g⁻¹ DW under the elicitation with 5.0 μmol l⁻¹ LLA and 5.0–20.0 μmol l⁻¹ α-LNA. Meanwhile, α-LNA was more effective than LLA for increasing biomass and ginsenoside production. The biomass of 11.1 g DW l⁻¹ and maximal total ginsenoside content of 7.9 mg g⁻¹ DW were achieved at 5 μmol l⁻¹ α-linolenic acid. The essential polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) fatty acids were accumulated in roots in response to elicitation while content of palmitic (C16:0) and oleic (C18:1) acids declined. The activities of SOD, G-POD and CAT were enhanced by two elicitors to similar extent while APX activity was preferably stimulated by α-LNA. Our results demonstrate that elicitation with α-linolenic acid stimulates production of biomass and secondary metabolites in bioreactor-cultured P. ginseng adventitious roots.     

The orientation-dependent expression of angiostatin-endostatin hybrid proteins and their characterization for the synergistic effects of antiangiogenesis

Two angiostatic fusion proteins (hAE and hEA) of human angiostatin (hAS) and endostatin (hES) proteins differed in tandem connection manner were constructed and evaluated for synergistic anti-angiogenic effects. The 65 kDa secreted fusion proteins from Pichia pastoris expression were verified by mass-spec analysis and western blotting assay. Luciferase reporter gene assay using VEGF promoter revealed that angiostatin-endostatin fusion protein (hAE) and its corresponding fusion gene delivery on Human Microvascular Endothelial Cells (HMEC-1) resulted in more potent synergistic anti-angiogenic effects than endostatin-angiostatin fusion protein (hEA). These facts suggest that the orientation of fusion genes between hAS and hES might be an important factor for developing therapeutic proteins.     

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.     

A novel WD40 protein,BnSWD1, is involved in salt stress in <Emphasis Type="Italic">Brassica napus</Emphasis>

Genes that are expressed early in specific response to high salinity conditions were isolated from rapeseed plant (Brassica napus L.) using an mRNA differential display method. Five PCR fragments (DD1–5) were isolated that were induced by, but showed different response kinetics to, 200 mM NaCl. Nucleotide sequence analysis and homology search revealed that the deduced amino sequences of three of the five cDNA fragments showed considerable similarity to those of β-mannosidase (DD1), tomato Pti-6 proteins (DD5), and the tobacco harpin-induced protein hin1 (DD4), respectively. In contrast, the remaining clones, DD3 and DD2, did not correspond to any substantial existing annotation. Using the DD3 fragment as a probe, we isolated a full-length cDNA clone from the cDNA library, which we termed BnSWD1 (Brassica napus salt responsive WD40 1). The predicted amino-acid sequence of BnSWD1 contains eight WD40 repeats and is conserved in all eukaryotes. Notably, the BnSWD1 gene is expressed at high levels under salt-stress conditions. Furthermore, we found that BnSWD1 was upregulated after treatment with abscisic acid, salicylic acid, and methyl jasmonate. Our study suggests that BnSWD1, which is a novel WD40 repeat-containing protein, has a function in salt-stress responses in plants, possibly via abscisic acid-dependent and/or -independent signaling pathways.     

Endoreduplication in <Emphasis Type="Italic">Phalaenopsis</Emphasis> is affected by light quality from light-emitting diodes during somatic embryogenesis

Endoreduplication is a developmental process that is unique to plants and occurs in all plants. The present study aimed to assess endoreduplication in various explant tissues and regenerated somatic embryos of Doritaenopsis. We further investigated the effects of light quality on endoreduplication and somatic embryo proliferation. To this end, we studied endoreduplication in leaves and root tips from regenerated plantlets and somatic embryos and in developing somatic embryos under 4 types of lighting conditions: red light, red + far-red light, red + blue light, and white light. We found that the degree of endoreduplication varied in different explants, and that the choice of explants used also influenced the ploidy levels of the newly regenerated somatic embryos. The DNA content of the leaf (2C–8C) was less than that of the root tip (2C–16C) and somatic embryo (2C–64C). In terms of light quality, the combination of red and far-red light produced the highest number of somatic embryos, while maintaining a low degree of endoreduplication. The data obtained indicate that this light combination stimulates somatic embryogenesis in Doritaenopsis and may exert some control on endoreduplication during cell division. These findings can be applied to achieve a reduction in somaclonal variations for the purpose of mass proliferation and genetic improvement.     

The need for validation of large administrative databases: Veterans Health Administration ICD-9CM coding of exudative age-related macular degeneration and ranibizumab usage

We performed a validation study by chart review of data for exudative age-related macular degeneration (eAMD) and, because of the Veterans Administration (VA) therapy policy, ranibizumab usage in the largest electronic medical record system in the USA. We reviewed 5,854 distinct patients who visited an ophthalmology clinic within VA Connecticut from January 2006-December 2008. We randomly selected 98 of 138 distinct eAMD patients and 265 of 5,588 non-eAMD patients who did not receive ranibizumab. International Classification of Diseases, Ninth Revision, Clinical Modification coding of eAMD had an excellent positive predictive value of 97.8% (95% confidence interval (CI), 93.5-99.4%). The national Decision Support System (DSS) had an excellent positive predictive value of 100% (95% CI, 79.9-100%) for ranibizumab. However, the negative predictive value of the DSS dispensed ranibizumab decreased to 67.5 (95% CI, 62.1-72.4) because of a change in the way local values were stored that led to errors. Therefore, validation of clinical information over time in large databases is necessary.     

Rapid upregulation ofDehyrin3 andDehydrin4 in response to dehydration is a characteristic of drought-tolerant genotypes in barley

The identification of molecular markers and marker-aided selection are essential to the efficient breeding of drought-tolerant plants. However, because that characteristic is controlled by many quantitative trait loci, such markers that can screen and trace desirable barley genotypes in a segregating population or germplasm have not yet been determined. Relative water content has been used to estimate drought tolerance in plants because it is highly correlated with the drought index of yield. To develop reliable gene-specific markers for identifying tolerant versus susceptible genotypes, we performed suppression subtractive hybridization to identify candidate genes. We used two domestic barley cultivars, one having the highest RWC (drought-tolerant ‘Chalbori’) and the other having the lowest (drought-susceptible ‘Daebaekbori’). In response to dehydration at the early seedling stage, rapid upregulation ofDehydrin3 (Dhn3) andDhn4 occurred in the drought-tolerant genotypes, but not in the susceptible ones. Similar results were obtained with mature plants growing under frequent drought stress in the greenhouse. In addition,Dhn3 andDhn4 conferred higher drought tolerance when they were over-expressed in transgenicArabidopsis. Thus, in addition to using assessments of RWC, we propose thatDhn3 andDhn4 expressions can serve as drought-induced gene-specific markers to determine drought-tolerant barley genotypes at the seedling stage.