Dong-Sun Lee

Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development

Human adipose tissue‐derived mesenchymal stem cells (hAT‐MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT‐MSC bioactive material (hAT‐MSC‐BM), and examined the effect of hAT‐MSC‐BM on porcine embryo development. hAT‐MSC‐BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT‐MSC‐BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT‐MSC culture medium group, the best results were from the group cultured with 10% hAT‐MSC‐BM. Mitochondrial activity was also higher in the 10% hAT‐MSC‐BM‐treated group. Moreover, the relative mRNA expression levels of development and anti‐apoptosis genes were significantly higher in the 10% hAT‐MSC‐BM‐treated group than in control, hAT‐MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT‐MSC‐BM starting on Day 4 also improved the development rate and the total cell number of in vitro‐fertilized embryos. This is the first report on the benefits of hAT‐MSC‐BM in a porcine embryo in vitro culture system. We conclude that hAT‐MSC‐BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro. Mol. Reprod. Dev. 80: 1035–1047, 2013.

Effect of plant growth regulator combination and culture period on in vitro regeneration of spinach (Spinacia oleracea L.)

The objective of this study was to develop an efficient system for the regeneration of spinach plants (Spinacia oleracea L.) by investigating the factors influencing callus and shoot induction. All plant growth regulator (PGR) combinations tested induced callus with high frequency (73–100xa0%), and the combination of 5xa0μM α-naphthaleneacetic acid (NAA), 10xa0μM 6-benzyladenine (BA) and 0.1xa0μM gibberellic acid (GA3) had the most significant effect on callus growth in term of weight (120.98xa0±xa022.56xa0mg). A high auxin-containing medium induced competent callus for shoot formation, while high cytokinin-containing media enhanced callus growth and made callus incompetent for shoot regeneration. Longer periods of callus induction in a high auxin-containing medium were required to form competent callus and led to a high regeneration capacity. The PGR combination shift from a high auxin to cytokinin ratio (ACR) to a low ACR resulted in highly efficient regeneration. Among the regeneration systems tested, the combination of 10xa0μM NAA and 0.3xa0μM GA3 for callus induction for 6xa0weeks followed by 2xa0μM NAA and 5xa0μM BA resulted in the highest plant regeneration frequency (83.33xa0±xa06.43xa0%) and the highest number of plantlets per explant (7.93xa0±xa01.24). Somatic embryos at cotyledonary stage and plantlets were transferred to PGR-free medium to establish whole plants. Regenerated female plants grew well to maturity in the greenhouse (77.17xa0±xa09.80xa0%) and produced seeds (175.21xa0±xa028.01 firm seeds per plant).

Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure

We developed an efficient Agrobacterium-mediated transformation protocol for spinach (Spinacia oleracea L.) that uses root-derived callus. Evaluation of this protocol was based on the systematic evaluation of factors that influence transformation efficiency. Four of the five factors that were tested significantly affected the transformation efficiency, including spinach cultivar, Agrobacterium tumefaciens strain and density, and the duration of co-cultivation. Transgenic spinach plants were generated based on optimized conditions, consisting of callus explants of the cultivar Gyeowoonae, A. tumefaciens strain EHA105 with OD600 of 0.2, a co-cultivation period of 4xa0d, and 100xa0μM acetosyringone supplemented in the inoculation and co-cultivation media. After co-cultivation with A. tumefaciens, explants were cultured in low-selective and then non-selective conditions to enhance the growth of putative transgenic explants. Visualization of the fluorescent marker, enhanced green fluorescent protein (EGFP), was used to select transgenic explants at several stages, including callus, somatic cotyledonary embryo, and plantlet. The best results for fluorescence visualization screening were obtained at the somatic cotyledonary embryo stage. On average, 24.96u2009±u20096.05% of the initial calli regenerated shoots that exhibited EGFP fluorescence. The putative transgenic plants were subjected to β-glucuronidase (GUS)-staining assay, phosphinothricin acetyltransferase (PAT) strip test, and molecular analyses to assess the transgene incorporation into plant genome and its expression. All EGFP-positive plants tested were confirmed to be transgenic by GUS-staining assay, PAT strip test, and molecular analyses. The transformation system described in this study could be a practical and powerful technique for functional genetic analysis and genetic modification of spinach.

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis. Graphical Abstract Plant regeneration of A. scaber via somatic embryogenesis

Fluctuation of 20-hydroxyecdysone in Individual Organs of Achyranthes japonica during Reproductive Growth Stage and Its Accumulation into Seed

To better understand 20-hydroxyecdysone (20E) fluctuation and accumulation in perennial plant, 20E concentration in individual organs of Achyranthes japonica during reproductive growth stage were analyzed by high performance liquid chromatography (HPLC). Concentrations of 20E in root and floral part were much higher than those in stem and leaf during reproductive growth stage and rapidly increased from flowering stage in August to seed-setting stage in October, and thereafter decreased at the stage of seed maturing in November. In contrast, the 20E concentrations in stem and leaf gradually decreased during reproductive growth. In the analysis of detailed fluctuation of 20E in floral part, the 20E concentration was highest in the seed at the early stage of seed development, compared to flower, peduncle, seed coat, and/or seed in other growth stages, and decreased during seed maturation. The accumulation of 20E in reproductive organs, especially seed and root, suggests that 20E has a defensive role for protection of especially newly developing organs against phytophagous insects.

Anti-viral activity of blue chanterelle (Polyozellus multiplex) that inhibits α-glucosidase

Blue chanterelle (Polyozellus multiplex), known as an edible mushroom, was extracted using methanol to screen on anti-viral agent. Syncytium formation in Newcastle disease virus (NDV)-infected baby hamster kidney (BHK) cell originates from the trafficking of viral glycoprotein into cell-surface. Blue chanterelle inhibited not only syncytium formation, but also trafficking of glycoprotein, hemagglutinin-neuramidase (HN), onto cell-surface. Viral glycoprotein is processed within the endoplasmic reticulum during routing to surface. Blue chanterelle extracts showed the inhibitory activities (IC50 10 μg/mL) against α-glucosidase. These results suggested that blue chanterelle extracts inhibited the cell-surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.

Catechol derived from aronia juice through lactic acid bacteria fermentation inhibits breast cancer stem cell formation via modulation Stat3/IL-6 signaling pathway

Cancer stem cells (CSCs) as a subpopulation of cancer cells are drug‐resistant and radiation‐resistant cancer cells to be responsible for tumor progress, maintenance and recurrence of cancer, and metastasis. This study isolated and investigated a new cancer stem cell (CSC) inhibitor derived from lactic acid fermentation products using culture broth with 2% aronia juice. The anti‐CSC activity of aronia‐cultured broth was significantly higher than that of the control. Activity‐guided fractionation and repeated chromatographic preparation led to the isolation of one compound. Using nuclear magnetic resonance and ESI mass spectrometry, we identified the isolated compound as catechol. In this study, we report that aronia‐fermented catechol has a novel inhibitory effect on human breast CSCs. Catechol inhibited breast cancer cell proliferation and mammosphere formation in a dose‐dependent manner. This compound reduced the CD44high/CD24low subpopulation, ALDH‐expressing cell population and the self‐renewal‐related genes nanog, sox2, and oct4. Catechol preferentially reduced mRNA transcripts and protein levels of Stat3 and did not induce c‐Myc degradation. These findings support the novel utilization of catechol for breast cancer therapy via the Stat3/IL‐6 signaling pathway. Our results suggest that catechol can be used for breast cancer therapy and that Stat3 expression is a marker of CSCs. Catechol inhibited Stat3 signaling by reducing Stat3 expression and secreted IL‐6, a CSC survival factor. These findings support the novel utilization of catechol for breast cancer therapy via Stat3/IL‐6 signaling.

Triterpene Acid (3-O-p-Coumaroyltormentic Acid) Isolated From Aronia Extracts Inhibits Breast Cancer Stem Cell Formation through Downregulation of c-Myc Protein

Cancer stem cells (CSCs) are drug-resistant and radiation-resistant cancer cells that are responsible for tumor progression and maintenance, cancer recurrence, and metastasis. Targeting breast CSCs with phytochemicals is a new paradigm for cancer prevention and treatment. In this study, activity-guided fractionation from mammosphere formation inhibition assays, repeated chromatographic preparations over silica gel, preparatory thin layer chromatography, and HPLC using aronia extracts led to the isolation of one compound. Using ¹H and 13C 2-dimensional nuclear magnetic resonance (NMR) as well as electrospray ionization (ESI) mass spectrometry, the isolated compound was identified as 3-O-p-coumaroyltormentic acid. This compound inhibits breast cancer cell proliferation and mammosphere formation in a dose-dependent manner and reduces the CD44high/CD24low subpopulation and aldehyde dehydrogenase (ALDH)-expressing cell population as well as the expression of the self-renewal-related genes CD44, SOX2, and OCT4.3-O-p-Coumaroyltormentic acid preferentially reduced the protein levels of c-Myc, which is a CSC survival factor, by inducing c-Myc degradation. These findings indicate the novel utilization of 3-O-p-coumaroyltormentic acid for breast cancer therapy via disruption of c-Myc protein, which is a CSC survival factor.

Proteasome Down-regulation is Partly Mediated by Slug/Snail2 in Hepatocarcinoma Cells

Snail family proteins (Snail1 and Slug/Snail2) are transcription factors that regulate transcription of molecules during epithelial-mesenchymal transition (EMT). Snail1/2 is known to bind to the E-box motif (CANNTG). The proteasome activity is decreased in EMT (Kim et al., 2011), and several E-box motifs are found in the promoters of genes coding for proteasome subunits. We used a new protein-binding microarray to specify the Slug/Snail2 binding sequence. Among 563 9-mer clusters, the motif CACCTGC yielded the highest P-value in the Wilcoxon-Mann-Whitney test. Within this motif, the A and T were absolutely required, and CC was preferred, but could be replaced by GG with little effect. In hepatocytes overexpressing Slug/Snail2, the 20S proteasome expression and proteasome activity were decreased partly due to the down-regulation of proteasome subunit beta type 2 (PSMB2) and PSMB3 transcription.

Production of recombinant miraculin protein using transgenic citrus cell suspension culture system

Miraculin gene containing the N-terminal signal peptide was introduced into navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) callus cells by Agrobacterum-mediated transformation. Transgenic somatic embryos were screened on the shoot induction medium containing 25 mg hygromycin L−1. Citrus callus cells were reproduced from the green color somatic embryos on the callus reproduction medium. The obtained transgenic cells were cultured in Murashige and Tucker’s liquid medium containing 50 g sucrose L−1 in a shaking incubator. Similar to the native miraculin, the secreted recombinant miraculin protein formed a disulfide-linked dimer and retained taste-modifying activity. The stability of recombinant protein expression was confirmed over nine generations of callus. This production system can be an excellent alternative for producing various recombinant proteins as well as miraculin.