Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 μM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 μM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 μM but decreased at 50 μM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O₂ ^•−) accumulation, and H₂O₂ content at 5 and 10 μM Cu-treated roots were not increased but strongly increased at 50 μM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 μM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 μM Cu, but both G-POD and CAT activities were inhibited at 50 μM Cu. Glutathione metabolism enzymes such as γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 μM Cu but were strongly inhibited at 50 μM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 μM Cu was associated to the strong induction of γ-glutamyltranspeptidase (γ-GGT) activity. These results indicate that plant could grow under Cu stress (5–25 μM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.     

Design,synthesis, and biological evaluation of novel diarylalkyl amides as TRPV1 antagonists

We have developed a new class of diarylalkyl amides as novel TRPV1 antagonists. They exhibited potent ⁴⁵Ca²⁺ uptake inhibitions in rat DRG neuron. In particular, the amide 59 was identified as a potent antagonist with IC₅₀ of 57 nM. The synthesis and structure–activity relationship of the diarylalkyl amides are also described.     

Nitric oxide retards xanthine oxidase-mediated superoxide anion generation in Phalaenopsis flower: an implication of NO in the senescence and oxidative stress regulation

Senescence is a developmentally regulated and highly ordered sequence of events. Senescence leads to abscission of plant organs and eventually leads to death of a plant or part of it. Present study revealed that Phalaenopsis flower undergo senescence due to over activation of O₂ ^·−generating xanthine oxidase (XO), which consequently increases the concentrations of O₂ ^·− leading to enhanced oxidative damage and disturbed cellular redox environment as indicated by increased lipid peroxidation and DHA/AsA + DHA ratio, respectively. While activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and non-specific peroxidase (POD) were enhanced in sepals and petals of old flower, activities of catalase (CAT) and glutathione reductase (GR) were decreased. Exogenous application of nitric oxide (NO) retarded H₂O₂-induced senescence of Phalaenopsis flower by downregulating activity of XO and concentrations of O₂ ^·−, H₂O₂ and malondialdehyde (MDA, an index of lipid peroxidation). Exogenous application of NO also downregulated SOD activity and upregulated antioxidant enzymes involved in the detoxification of H₂O₂ (CAT and APX), and in the regulation of redox couples viz, monodehydroascorbate reductase (MDHAR) and GR, together with the modulation in non-protein thiol status and DHA/AsA + DHA ratio.     

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.