• Bulletin of the Korean Chemical Society
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• v.19 no.8
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• pp.836-840
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• 1998

The intrinsic and equilibrium isotope effects on the 13C NMR chemical shift of the cyclooctanone-2-D were investigated. Equilibrium constants and changes in the free energies, enthalpy, entropy, which are derived from the temperature dependence of the isotope shifts, are reported for this isotopomer.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.5
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• pp.237-251
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• 2002

The recent progress on metabolic systems engineering was reviewed based on our recent research results in terms of (1) metabolic signal flow diagram approach, (2) metabolic flux analysis (MFA) in particular with intracellular isotopomer distribution using NMR and/or GC-MS, (3) synthesis and optimization of metabolic flux distribution (MFD), (4) modification of MFD by gene manipulation and by controlling culture environment, (5) metabolic control analysis (MCA), (6) design of metabolic regulation structure, and (7) identification of unknown pathways with isotope tracing by NMR. The main characteristics of metabolic engineering is to treat metabolism as a network or entirety instead of individual reactions. The applications were made for poly-3-hydroxybutyrate (PHB) production using Ralstonia eutropha and recombinant Escherichia coli, lactate production by recombinant Saccharomyces cerevisiae, pyruvate production by vitamin auxotrophic yeast Toluropsis glabrata, lysine production using Corynebacterium glutamicum, and energetic analysis of photosynthesic microorganisms such as Cyanobateria. The characteristics of each approach were reviewed with their applications. The approach based on isotope labeling experiments gives reliable and quantitative results for metabolic flux analysis. It should be recognized that the next stage should be toward the investigation of metabolic flux analysis with gene and protein expressions to uncover the metabolic regulation in relation to genetic modification and/ or the change in the culture condition.

• Analytical Science and Technology
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• v.7 no.2
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• pp.213-224
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• 1994

Several isotopomers of cyclooctanone were prepared by selective deuterium substitution. Intrinsic isotope effects on $^{13}C$ NMR chemical shifts of these isotopomers were investigated systematically at low temperature. These istope effects were discussed in relation to the preferred boat-chair conformation of cyclooctanone. Deuterium isotope effects on NMR chemical shifts have been known for a long time. Especially in a conformationally mobile molecule, isotope perturbation could affect NMR signals through a combination of isotope effects on equilibria and intrinsic effects. The distinction between intrinsic and nonintrinsic effects is quite difficult at ambient temperature due to involvement of both equilibrium and intrinsic isotope effects. However if equilibria between possible conformers of cyclooctanone are slowed down enough on the NMR time scale by lowering temperature, it should be possible to measure intrinsic isotope shifts from the separated signals at low temperature. $^{13}C$ NMR has been successfully utilized in the study on molecular conformation in solution when one deals with stable conformers or molecules were rapid interconversion occurs at ambient temperature. The study of dynamic processes in general requires analysis of spectra at several temperature. Anet et al. did $^1H$ NMR study of cyclooctanone at low temperature to freeze out a stable conformation, but were not able initially to deduce which conformation was stable because of the complexity of alkyl region in the $^1H$ NMR spectrum. They also reported the $^1H$ and $^{13}C$ NMR spectra of the $C_9-C_{16}$ cycloalkanones with changing temperature from $-80^{\circ}C$ to $-170^{\circ}C$, but they did not report a variable temperature $^{13}C$ NMR study of cyclooctanone. For the analysis of the intrinsic isotope effect with relation to cylooctanone conformation, $^{13}C$ NMR spectra are obtained in the present work at low temperatures (up to $-150^{\circ}C$) in order to find the chemical shifts at the temperature at which the dynamic process can be "frozen-out" on the NMR time scale and cyclooctanone can be observed as a stable conformation. Both the ring inversion and pseudorotational processes must be "frozen-out" in order to see separate resonances for all eight carbons in cyclooctanone. In contrast to $^1H$ spectra, slowing down just the ring inversion process has no apparent effects on the $^{13}C$ spectra because exchange of environments within the pairs of methylene carbons can still occur by the pseudorotational process. Several isotopomers of cyclooctanone were prepared by selective deuterium substitution (fig. 1) : complete deuterium labeling at C-2 and C-8 positions gave cyclooctanone-2, 2, 8, $8-D_4$ : complete labeling at C-2 and C-7 positions afforded the 2, 2, 7, $7-D_4$ isotopomer : di-deuteration at C-3 gave the 3, $3-D_2$ isotopomer : mono-deuteration provided cyclooctanone-2-D, 4-D and 5-D isotopomers : and partial deuteration on the C-2 and C-8 position, with a chiral and difunctional case catalyst, gave the trans-2, $8-D_2$ isotopomer. These isotopomer were investigated systematically in relation with cyclooctanone conformation and intrinsic isotope effects on $^{13}C$ NMR chemical shifts at low temperature. The determination of the intrinsic effects could help in the analysis of the more complex effects at higher temperature. For quantitative analysis of intrinsic isotope effects, the $^{13}C$ NMR spectrum has been obtained for a mixture of the labeled and unlabeled compounds because the signal separations are very small.

• Preventive Nutrition and Food Science
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• v.10 no.1
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• pp.40-45
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• 2005

Age-related changes in bone metabolism are well established by biochemical markers of bone matrix in serum and urine, but analysis of the residual bone matrix, which is still turning over, has not been investigated. In the present study, we measured in vivo rates of bone protein synthesis using a precursor-product method based on the exchange of ²H from ²H₂O into amino acids. Four percent ²H₂O was administered to mice in drinking water after intraperitonial (i.p) bolus injection of 99.9% ²H₂O. Mice were divided into the two groups: growing young mice were administered 4% ²H₂O for 12 weeks after an i.p bolus injection at 5 week of age, whereas weight stable adult mice started drinking 4% ²H₂O 8 weeks later than the growing group and continued 4% ²H₂O drinking for 8 weeks. Mass isotopomer abundance in alanine from bone protein was analyzed by gas chromatography/mass spectrometry. Body ²H₂O enrichments were in the range of 1.88-2.41% over the labeling period. The fractional synthesis rates (ks) of bone protein were 2.000±0.071%/d for growing mice and 0.243±0.014%/d for adult mice. These results demonstrate that the bone protein synthesis rate decreases with age and present direct evidence of age-related changes in bone protein synthesis.

• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1174-1179
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• 2006

The wild-type Corynebacterium glutamicum ATIC 13032 and Corynebacterium glutamicum ATTC 13032 lysC S301Y, exhibiting a deregulated aspartokinase, were compared concerning growth, lysine production, and intracellular carbon fluxes. Both strains differ by only one single nucleotide over the whole genome. In comparison to the wild-type, the mutant showed significant production of lysine with a molar yield of 0.087 mol (mol glucose$^{-1}$) whereas the biomass yield was reduced. The deregulation of aspartokinase further led to a global rearrangement of carbon flux throughout the whole central metabolism. This involved an increased flux through the pentose phosphate pathway (PPP) and an increased flux through anaplerosis. Because of this, the mutant revealed an enhanced supply of NADPH and oxaloacetate required for lysine biosynthesis. Additionally, the lumped flux through phosphoenolpyruvate carboxykinase and malic enzyme, withdrawing oxaloacetate back to the glycolysis and therefore detrimental for lysine production, was increased. The reason for this might be a contribution of malic enzyme to NADPH supply in the mutant in the mutant. The observed complex changes are remarkable, because they are due to the minimum genetic modification possible, the exchange of only one single nucleotide.

• The Bulletin of The Korean Astronomical Society
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• v.39 no.2
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• pp.75.2-75.2
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• 2014

CS molecule as an important tracer for studying inward motions in dense cores is known to be adsorbed onto dusts in cold (T~10K) dense cores, resulting in its significant depletion in the central region of the cores which may hamper a proper study of kinematics stage of star formation. In this study we choose five 'evolved' dense starless cores, L1544, L1552, L1689B, L694-2 and L1197, to investigate how depletion of CS molecule is significant and how the molecule differentiates depending on the evolutional status of the dense cores, by using a rare isotopomer C34S. We performed mapping observations in C34S (J=2-1) and N2H+ (J=1-0) with Nobeyama 45 m telescope, and compared $850{\mu}m$ continuum data as a reference of the density distribution of the dense cores. Our data confirm the claim that CS molecule generally depletes out in the central region in dense starless cores, while N2H+ keeps abundant as they get evolved. All of integrated intensity maps show 'semi-ring-like' depletion holes in CS, and all of abundance radial profiles show decrease toward center. The CS depletion and molecular chemical differentiation seems to depend on the evolutional status in dense cores. The evolved cores shows low abundance at both central and outer regions, implying that in the case of highly evolved cores CS freeze-out occurs over the most area of the cores.

• BMB Reports
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• v.45 no.7
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• pp.396-401
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• 2012

Overnutrition is one of the major causes of non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by an accumulation of lipids (triglycerides) in hepatocytes and is often accompanied by high plasma levels of free fatty acids (FFA). In this study, we compared the energy metabolism in acute steatotic and non-steatotic primary mouse hepatocytes. Acute steatosis was induced by pre-incubation with high concentrations of oleate and palmitate. Labeling experiments were conducted using [$U-^{13}C_5$,$U-^{15}N_2$] glutamine. Metabolite concentrations and mass isotopomer distributions of intracellular metabolites were measured and applied for metabolic flux estimation using transient $^{13}C$ metabolic flux analysis. FFAs were efficiently taken up and almost completely incorporated into triglycerides (TAGs). In spite of high FFA uptake rates and the high synthesis rate of TAGs, central energy metabolism was not significantly changed in acute steatotic cells. Fatty acid ${\beta}$-oxidation does not significantly contribute to the detoxification of FFAs under the applied conditions.

• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.23-36
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• 2009

In the present work, the metabolic network of primary metabolism of the slow-growing myxobacterium Sorangium cellulosum was reconstructed from the annotated genome sequence of the type strain So ce56. During growth on glucose as the carbon source and asparagine as the nitrogen source, So ce56 showed a very low growth rate of $0.23\;d^{-1}$, equivalent to a doubling time of 3 days. Based on a complete stoichiometric and isotopomer model of the central metabolism, $^{13}C$ metabolic flux analysis was carried out for growth with glucose as carbon and asparagine as nitrogen sources. Normalized to the uptake flux for glucose (100%), cells recruited glycolysis (51%) and the pentose phosphate pathway (48%) as major catabolic pathways. The Entner-Doudoroff pathway and glyoxylate shunt were not active. A high flux through the TCA cycle (118%) enabled a strong formation of ATP, but cells revealed a rather low yield for biomass. Inspection of fluxes linked to energy metabolism revealed that S. cellulosum utilized only 10% of the ATP formed for growth, whereas 90% is required for maintenance. This explains the apparent discrepancy between the relatively low biomass yield and the high flux through the energy-delivering TCA cycle. The total flux of NADPH supply (216%) was higher than the demand for anabolism (156%), indicating additional reactions for balancing of NADPH. The cells further exhibited a highly active metabolic cycle, interconverting $C_3$ and $C_4$ metabolites of glycolysis and the TCA cycle. The present work provides the first insight into fluxes of the primary metabolism of myxobacteria, especially for future investigation on the supply of cofactors, building blocks, and energy in myxobacteria, producing natural compounds of biotechnological interest.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.8
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• pp.1390-1393
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• 2003

Measuring in vivo rate of bone collagen synthesis has so far been technically difficult and often subject to quite large errors. In the present study, bone collagen synthesis rate was measured using a precursor-product method, based on the exchange of $^2$$H_2O$ into amino acids. Mass isotopomer abundance in hydroxyproline from bone collagen was analyzed by gas chromatography/mass spectrometry. The $^2$$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 9 weeks. Body $^2$$H_2O$ enrichments were stable at 2.7 ∼ 3.0% over labeling Period. In growing rats, the fractional synthesis rate ( $k_{s}$) of bone collagen was 0.066 $\pm$ 0.049 w $k^{-1}$ . The unique features of stable $^2$$H_2O$ pools and label incorporation allowed the precursor-product approach to be used for measuring bone collagen synthesis rate..