Somi K. Cho

Simple, Efficient, and Reproducible Gene Transfection of Mouse Embryonic Stem Cells by Magnetofection

Embryonic stem (ES) cells are recognized as an excellent cell culture model for studying developmental mechanisms and their therapeutic modulations. The aim of this work was to define whether using magnetofection was an efficient way to manipulate stem cells genetically without adversely affecting their proliferation or self-renewal capacity. We compared our magnetofection results to those of a conservative method using FuGENE 6. Using enhanced green fluorescent protein (eGFP) as a reporter gene in D3 mouse ES (mES) cells, we found that magnetofection gave a significantly higher efficiency (45%) of gene delivery in stem cells than did the FuGENE 6 method (15%), whereas both demonstrated efficient transfection in NIH-3T3 cells (60%). Although the transfected D3 (D3-eGFP) mES cells had undergone a large number of passages (>50), a high percentage of cells retained ES markers such as Oct-4 and stage-specific embryonic antigen-1 (SSEA-1). They also retained the ability to form embryoid bodies and differentiated in vitro into cells of the three germ layers. eGFP expression was sustained during stem cell proliferation and differentiation. This is the first transfection report using magnetofection in ES cells. On the basis of our results, we conclude that magnetofection is an efficient and reliable method for the introduction of foreign DNA into mouse ES cells and may become the method of choice.

Classification and prediction of free-radical scavenging activities of dangyuja (Citrus grandis Osbeck) fruit extracts using 1H NMR spectroscopy and multivariate statistical analysis

Different parts of dangyuja (Citrus grandis Osbeck) fruits at different maturation stages were classified using a (1)H NMR-based metabolomic technique. Principal components analysis allowed the clear separation of fractions extracted with 50% methanol of different parts of dangyuja fruits at different maturation stages by combining principal components PC1 and PC2, which together accounted for 80.4% of the variance. A loading-plot analysis revealed that sucrose, glucose, oxaloacetic acid and citric acid were dominant in mature flesh, while naringin, tyramine, proline and alanine were dominant in immature fruit samples. Projections to latent structures using a partial least squares (PLS) model were used to predict the free-radical scavenging activities (FRSA) of dangyuja fruit extracts based on their (1)H NMR spectra. The present study suggests the usefulness of combining (1)H NMR spectroscopy with multivariate statistical analysis for discriminating dangyuja fruit samples, and predicting the FRSA of different parts of dangyuja fruit samples at different stages of maturation.

Metabolic Profiling and Predicting the Free Radical Scavenging Activity of Guava (Psidium guajava L.) Leaves According to Harvest Time by 1H-Nuclear Magnetic Resonance Spectroscopy

Guava leaves were classified and the free radical scavenging activity (FRSA) evaluated according to different harvest times by using the 1H-NMR-based metabolomic technique. A principal component analysis (PCA) of 1H-NMR data from the guava leaves provided clear clusters according to the harvesting time. A partial least squares (PLS) analysis indicated a correlation between the metabolic profile and FRSA. FRSA levels of the guava leaves harvested during May and August were high, and those leaves contained higher amounts of 3-hydroxybutyric acid, acetic acid, glutamic acid, asparagine, citric acid, malonic acid, trans-aconitic acid, ascorbic acid, maleic acid, cis-aconitic acid, epicatechin, protocatechuic acid, and xanthine than the leaves harvested during October and December. Epicatechin and protocatechuic acid among those compounds seem to have enhanced FRSA of the guava leaf samples harvested in May and August. A PLS regression model was established to predict guava leaf FRSA at different harvesting times by using a 1H-NMR data set. The predictability of the PLS model was then tested by internal and external validation. The results of this study indicate that 1H-NMR-based metabolomic data could usefully characterize guava leaves according to their time of harvesting.

Distribution and Biosynthesis of 20-Hydroxyecdysone in Plants of Achyranthes japonica Nakai

There is increasing interest in phytoecdysteroids (PEs) because of their potential role in plant defense against insects. To understand the mechanism regulating their levels in plants, the fluctuation, distribution, and biosynthesis of PE 20-hydroxyecdysone (20E) examined in Achyranthes japonica. The total amount of 20E per individual plant initially remained at a constant level, and increased markedly after the first leaf pair (LP) stage, while the concentration of 20E in a given plant decreased rapidly during vegetative growth. In addition, the incorporation of [2-14C]-mevalonic acid into 20E did not differ significantly depending on plant organs and developmental stages, suggesting that biosynthesis of 20E is not restricted to particular organs or growth stages.

Nonactin hinders intracellular glycosylation in virus-infected baby hamster kidney cells

Potent antiviral agents hinder virus-infected cell machinery, leading to rescue from viral damage. In this study, we aimed to identify selective intracellular glycosylation inhibitor(s) that do not suppress glycoprotein synthesis. Our results showed that nonactin is a potent inhibitor of intracellular glycosylation. First, we examined the effects of nonactin on syncytium formation and cytopathic activity in virus-infected baby hamster kidney cells. Nonactin effectively inhibited syncytium formation in a concentration-dependent manner, and infectious virus production was markedly reduced. However, glycoprotein synthesis was not affected. In the presence of 5 µg/ml nonactin, we observed the intracellular accumulation of vesicular stomatitis virus-G protein as well as syncytium formation, but no significant effects on Newcastle disease virus-hemagglutinin-neuramidase glycoprotein synthesis. Our results collectively indicate that nonactin potentially inhibits glycosylation by acting as a suppressor of intracellular glycosylation trafficking.

Ameliorating effects of Mango (Mangifera indica L.) fruit on plasma ethanol level in a mouse model assessed with 1H-NMR based metabolic profiling

The ameliorating effects of Mango (Mangifera indica L.) flesh and peel samples on plasma ethanol level were investigated using a mouse model. Mango fruit samples remarkably decreased mouse plasma ethanol levels and increased the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase. The 1H-NMR-based metabolomic technique was employed to investigate the differences in metabolic profiles of mango fruits, and mouse plasma samples fed with mango fruit samples. The partial least squares-discriminate analysis of 1H-NMR spectral data of mouse plasma demonstrated that there were clear separations among plasma samples from mice fed with buffer, mango flesh and peel. A loading plot demonstrated that metabolites from mango fruit, such as fructose and aspartate, might stimulate alcohol degradation enzymes. This study suggests that mango flesh and peel could be used as resources for functional foods intended to decrease plasma ethanol level after ethanol uptake.

Erratum to: Trafficking inhibition of bruceanol B as a radical-producing antibiotic

Acknowledgments. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) (2010-0477), the Gene and Material Bank for Citrus Breeding at Jeju National University (KRF-2007-412-J05502), Priority Research Centers Program (2010-0029630), and Mid-career Researcher Program (2010-0027752) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.