• Journal of Microbiology and Biotechnology
• /
• v.20 no.8
• /
• pp.1196-1203
• /
• 2010

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-$CH_3$ group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the ${\Delta}$metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.

• Journal of the Korean Magnetic Resonance Society
• /
• v.16 no.1
• /
• pp.34-45
• /
• 2012

Melanocortin-4 receptor (MC4R) subtype is associated with obese humans. Especially, in a patient with severe early-onset obesity, novel heterozygous mutation in the MC4R gene was detected, resulting in an exchange of aspartic acid to asparagine in $90^{th}$ amino acid residue located in the predicted second trans-membrane domain (TM2). Mutations in the melanocortin-4 receptor (MC4R) gene are the most frequent monogenic causes of severe obesity which have been described as heterozygous with loss of function. In order to compare structure difference between MC4R wild type (MC4R-TM2-wt) and mutant (MC4R-TM2-D90N), we designed both MC4R-TM2-wt and MC4R-TM2-D90N construct in pET 21b vector. In this study, we optimized high-yield purification procedure for recombinant TM2-D90N. Eluted recombinant protein was resolubilized under urea condition for thrombin cleavage reaction and we conducted the high-performance liquid chromatography (HPLC) with reverse phase column under 1% acetonitrile, 0.01% TFA buffer solution. The molecular size of purified target peptide was confirmed by Tricine-SDS page analysis. To characterize MC4R-TM2-D90N, we have performed $^{15}N$-isotope labeling of peptide using M9 media and purified labeled target peptide for hetero-nuclear NMR spectroscopy.

• Journal of Radiopharmaceuticals and Molecular Probes
• /
• v.1 no.1
• /
• pp.23-30
• /
• 2015

Radioisotopes emitting low-range highly ionizing radiation such as ${\beta}$-particles are of increasing significance in internal radiotherapy. Among the ${\beta}$-particle emitting radioisotopes, $^{67}Cu$ is an attractive radioisotope for various nuclear medicine applications due to its medium energy ${\beta}$-particle, gamma emissions, and 61.83-hour half-life, which can also be used with $^{64}Cu$ for PET imaging. The production and application of the ${\beta}$-emitting radioisotope $^{67}Cu$ for therapeutic radiopharmaceutical are outlined, and different production routes are discussed. A survey of copper chelators used for antibody labeling is provided. It has been produced via proton, alpha, neutron, and gamma irradiations followed by solvent extraction, ion exchange, electrodeposition. Clinical studies using $^{67}Cu$-labelled antibodies in lymphoma, colon carcinoma and bladder cancer patients are reviewed. Widespread use of this isotope for clinical studies and preliminary treatments has been limited by unreliable supplies, cost, and difficulty in obtaining therapeutic quantities.

• Mass Spectrometry Letters
• /
• v.3 no.4
• /
• pp.93-100
• /
• 2012

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. In order to enhance the understanding of molecular dynamics for biological process in detail, it is necessary to develop sensitive and comprehensive analytical methods for the determination of protein phosphorylation. Neural stem cells hold great promise for neural repair following an injury or disease. In this study, we made differentiated oligodendrocytes from human neural stem cells using over-expression of olig2 gene. We confirmed using quantitative phosphoproteome analysis approach that combines stable isotope labeling by amino acids in cell culture (SILAC) and $TiO_2$ micro-column for phosphopeptide enrichment with $MS^2$ and $MS^3$ mass spectrometry. We detected 275 phosphopeptides which were modulated at least 2-fold between human neural stem cells and oligodendrocytes. Among them, 23 phosphoproteins were up-regulated in oligodendrocytes and 79 phosphoproteins were up-regulated in F3 cells.

• Journal of Ginseng Research
• /
• v.40 no.3
• /
• pp.278-284
• /
• 2016

Background: The ginsenoside Rb1 (Rb1) is the most abundant compound in the root of Panax ginseng. Recent studies have shown that Rb1 has a neuroprotective effect. However, the mechanisms underlying this effect are still unknown. Methods: We used stable isotope labeling with amino acids in cell culture, combined with quantitative mass spectrometry, to explore a potential protective mechanism of Rb1 in ${\beta}$-amyloid-treated neuronal cells. Results: A total of 1,231 proteins were commonly identified from three replicate experiments. Among these, 40 proteins were significantly changed in response to Rb1 pretreatment in ${\beta}$-amyloid-treated neuronal cells. Analysis of the functional enrichments and protein interactions of altered proteins revealed that actin cytoskeleton proteins might be linked to the regulatory mechanisms of Rb1. The CAP1, CAPZB, TOMM40, and DSTN proteins showed potential as molecular target proteins for the functional contribution of Rb1 in Alzheimer's disease (AD). Conclusion: Our proteomic data may provide new insights into the protective mechanisms of Rb1 in AD.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.33 no.1
• /
• pp.218-221
• /
• 2004

This study was performed to investigate the effect of rosiglitazone, a new antidiabetic agent, on in vivo synthesis of bone collagen. The mice were divided into low-fat diet group (LF), high-fat diet group (HF), and high-fat diet with rosiglitazone (6.3 $\mu\textrm{g}$/kcal diet) group (HF-Rosi), The synthesis of bone collagen was measured by stable isotope-mass spectrometric technique using $^2$$H_2O$ as a tracer. The $^2$$H_2O$ labeling protocol consisted of an initial intraperitoneal injection of 99.9% $^2$$H_2O$, to achieve approximately 2.5% body water enrichment followed by administration of 4％ $^2$$H_2O$ in drinking water for 3 weeks. Although body weight gain and daily diet intake were not significantly different between groups, HF-Rosi had slightly higher body weight gain and daily diet intake than LF and HF. In addition, HF-Rosi showed significantly higher body fat content than LF and HF. Bone collagen synthesis was reduced in HF than LF and further decreased by the treatment of rosiglitazone. These results suggest rosiglitazone affect body fat content and bone turnover in mice.

• Korean Journal of Ecology and Environment
• /
• v.52 no.3
• /
• pp.202-209
• /
• 2019

The primary productivity and production rate of photosynthetic pigment of periphyton and phytoplankton were estimated using a $^{13}C$ stable labeling technique in May 2011, in the waterfront of Lake Paldang. Primary productivity of periphyton ($28.15mgC\;m^{-2}\;d^{-1}$) was higher than phytoplankton production ($0.14mgC\;m^{-2}\;d^{-1}$). The net production rates of photosynthetic pigments(Chl a and Fucoxanthin) of periphyton were $2.53ngC\;m^{-2}\;d^{-1}$ and $0.12ngC\;m^{-2}\;d^{-1}$, respectively. On the other hand, the net production rate of pigments on phytoplankton (Chl a : $0.023ngC\;m^{-2}\;d^{-1}$, Fucoxanthin: $0.002ngC\;m^{-2}\;d^{-1}$) was lower than that of periphyton. Specific production rates of individual pigments of phytoplankton to the total primary productivity indicate the predominance of diatom (Fucoxanthin) species in phytoplankton assemblage in Lake Paldang. The net individual production rate of pigments by $^{13}C$ tracer was a useful tool to estimate the contribution of each phytoplankton class for total primary productivity, and it is possible to calculate the seasonal contribution of each phytoplankton class to the total primary productivity in the aquatic ecosystems. This study is the first report on photosynthetic pigment production rates of periphyton and phytoplankton.