• Title/Summary/Keyword: in-cell NMR

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Tubular Type Direct Methanol Fuel Cell for in situ NMR Diagnosis (In Situ NMR 진단용 원통형 직접 메탄올 연료전지)

  • Joh, Han-Ik;Um, Myung-Sup;Han, Kee-Sung;Han, Oc-Hee;Ha, Heung-Yong;Kim, Soo-Kil
    • Journal of the Korean Electrochemical Society
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    • v.12 no.4
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    • pp.329-334
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    • 2009
  • This study is to develop a fuel cell system applicable to an in situ NMR (Nuclear magnetic resonance) diagnosis. The in situ NMR can be used in real time monitoring of various reactions occurring in the fuel cell, such as oxidation of fuel, reduction of oxygen, transport phenomena, and component degradation. The fuel cell for this purpose is, however, to be operated in a specifically designed tubular shape toroid cavity detector (TCD), which constrains the fuel cell to have a tubular shape. This may cause difficulties in effective mass transport of reactants/products and uniform distribution of assembly pressure. Therefore, a new flow field designed in a particular way is necessary to enhance the mass transport in the tubular fuel cell. In this study, a tubular-shaped close-type flow field made of non-magnetic material is developed. With this flow field, oxygen is effectively delivered to the cathode surface and the produced water is readily removed from the membrane-electrode assembly to prevent flooding. The resulting DMFC (direct methanol fuel cell) outperforms the open-type flow field and exhibits $36\;mW/cm^2$ even at room temperature.

In-cell nuclear magnetic resonance spectroscopy for studying intermolecular interactions

  • Sugiki, Toshihiko;Lin, Yuxi;Lee, Young-Ho
    • Journal of the Korean Magnetic Resonance Society
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    • v.23 no.1
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    • pp.33-39
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    • 2019
  • Studies on the interactions of proteins with partner molecules at the atomic resolution are essential for understanding the biological function of proteins in cells and for developing drug molecules. Solution NMR spectroscopy has shown remarkably useful capability for investigating properties on the weak to strong intermolecular interactions in both diluted and crowded solution such as cell lysates. Of note, the state-of-the-art in-cell NMR method has made it possible to obtain atomistic information on natures of intermolecular interactions between target proteins with partner molecules in living cells. In this mini-review, we comprehensively describe the several technological advances and developments in the in-cell NMR spectroscopy.

Solution Structure of the Cytoplasmic Domain of Syndecan-3 by Two-dimensional NMR Spectroscopy

  • Yeo, In-Young;Koo, Bon-Kyung;Oh, Eok-Soo;Han, Inn-Oc;Lee, Weon-Tae
    • Bulletin of the Korean Chemical Society
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    • v.29 no.5
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    • pp.1013-1017
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    • 2008
  • Syndecan-3 is a cell-surface heparan sulfate proteoglycan, which performs a variety of functions during cell adhension process. It is also a coreceptor for growth factor, mediating cell-cell and cell-matrix interaction. Syndecan-3 contains a cytoplasmic domain potentially associated with the cytoskeleton. Syndecan-3 is specifically expressed in neuron cell and has related to neuron cell differentiation and development of actin filament in cell migration. Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains. And that region of syndecan-3 may modulate the interactions of the conserved C1 regions of the cytoplasmic domains by tyrosine phosphorylation. Cytoplasmic domain of syndecan-3 has been synthesized for NMR structural studies. The solution structure of syndecan-3 cytoplasmic domain has been determined by two-dimensional NMR spectroscopy and simulated-annealing calculation. The cytoplasmic domain of the syndecan proteins has a tendency to form a dimmer conformation with a central cavity, however, that of syndecan-3 demonstrated a monomer conformation with a flexible region near C-terminus. The structural information might add knowledge about the structure-function relationships among syndecan proteins.

In Vitro Detection of Apoptosis in Human Promyleoytic Leukemia HL-60 Cells by $^{1}H$-NMR

  • Lee, Chul-Hoon;Lee, Min-A.;Cho, Youl-Hee;Lim, Hae-Young;Jung, Ji-Hyun;Kim, Kyung H.;Lim, Yoong-Ho
    • Journal of Microbiology and Biotechnology
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    • v.11 no.3
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    • pp.539-542
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    • 2001
  • $^1H$-NMR spectroscopy was used to detect apoptosis in HL-60 cells in vitro. The relationship between cell apoptosis and NMR data was validated by the flow cytometry assay. To evaluate the NMR apoptosis results, the ratio of methylene and methyl groups caused by lipids was used. In addition, an identical analysis was applied to HepG2 cells. Detection of apoptotic cell death by NMR spectroscopy was oserved.

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Establishment of a Simple and Rapid Method for Quantitative Determination of -Aminobutyric Acid Using 1H NMR and Production of γ-Aminobutyric Acid in Cell Suspension Cultures of Coriandrum sativum L. (1H NMR에 의한 γ-Aminobutyric Acid의 간단하고 신속한 정량분석법 확립과 고수 (Coriandrum sativum L.) 현탁배양세포로부터 γ-Aminobutyric Acid의 생산)

  • Kim, Suk-Weon;Liu, Jang-R.
    • Journal of Plant Biotechnology
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    • v.34 no.3
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    • pp.207-212
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    • 2007
  • This study describes a simple and rapid method for quantitative determination of $\gamma$-aminobutyric acid (GABA) using $^1H$ NMR spectroscopy from whole cell extracts of plant suspension cultures. When 9 cell lines derived from 8 species of higher plants maintained in liquid Marashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) were subjected to $^1H$ NMR, a cell line of Coriandrum sativum L. exhibited the highest level of GABA. The level reached up to 16.9 mg/dry wt when cells were cultured in MS medium supplemented with 0.5 mg/L 2,4-D after 3 weeks of incubation. The method for quantitative determination of GABA using $^1H$ NMR established in this study could be applied to high-throughput screening of various plant resources for GABA production and the cell suspension culture system of C. sativum could be further developed for commercial production of GABA.

Heteronuclear NMR studies on 44 kDa dimer, syndesmos

  • Kim, Heeyoun;Lee, Inhwan;Han, Jeongmin;Cheong, Hae-kap;Kim, Eunhee;Lee, Weontae
    • Journal of the Korean Magnetic Resonance Society
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    • v.19 no.2
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    • pp.83-87
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    • 2015
  • Syndesmos, which is co-localized with syndecan-4 cytoplasmic domain ($Syn4^{cyto}$) in focal contacts, interacts with various cell adhesion adaptor proteins including $Syn4^{cyto}$ to control cell signaling. Syndesmos consists of 211 amino acids and it exists as a dimer (44kDa) in solution. Recently, we have determined the structure of syndesmos by x-ray crystallography, however, dynamics related to syndecan binding still remain elusive. In this report, we performed NMR experiments to acquire biochemical and structural information of syndesmos. Based on a series of three-dimensional triple resonance experiments on a $^{13}C/^{15}N/^2H$ labeled protein, NMR spectra were obtained with well dispersed and homogeneous NMR data. We present the sequence specific backbone assignment of syndesmos and assigned NMR data with combination structural information can be directly used for the studies on interaction with $Syn4^{cyto}$ and other binding molecules.

NMR Structure of Syndecan-4L reveals structural requirement for PKC signalling

  • Koo, Bon-Kyoung;Joon Shin;Oh, Eok-Soo;Lee, Weontae
    • Proceedings of the Korean Magnetic Resonance Society Conference
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    • 2002.08a
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    • pp.90-90
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    • 2002
  • Syndecans, transmembrane heparan sulfate proteoglycans, are coreceptors with integrin in cell adhesion process. It forms a ternary signaling complex with protein kinase C and phosphatidylinositol 4,5 bisphosphate (PIP2) for integrin signaling. NMR data indicates that cytoplasmic domain of syndecan-4 (4L) undergoes a conformational transition in the presence of PIP2, forming oligomeric conformation. The structure based on NMR data demonstrated that syndecan-4L itself forms a compact intertwined symmetric dimer with an unusual clamp shape for residues Leu$^{186}$ -Ala$^{195}$ . The molecular surface of the syndecan-4L dimer is highly positively charged. In addition, no inter-subunit NOEs in membrane proximal amino acid resides (Cl region) has been observed, demonstrating that the Cl region is mostly unstructured in syndecan-4L dimmer. However, the complex structure in the presence of PIP2 induced a high order multimeric conformation in solution. In addition, phosphorylation of cytoplasmic domain induces conformational change of syndecan-4, resulting inhibition of PKC signaling. The NMR structural data strongly suggest that PIP2 promotes oligomerization of syndecan-4 cytoplasmic domain for PKC activation and further induces structural reorganization of syndecan for mediating signaling network in cell adhesion procedure.

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Characterization of C-P Lyase gene cluster by in vivo $^{31}$ P-NMR spectroscopy

  • Lee, Ki-Sung;Kwak, In-Young
    • Journal of Microbiology
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    • v.33 no.4
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    • pp.328-333
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    • 1995
  • $\^$31/ P-NMR experiment was performed to detect phophonates (Pn) utilization and degradation in the several different C-P lyase mutants of E. coli and in E. aerogenes and the recombinants. The relative peak intensity (RPI) for the standard samples of 0.5 mM methylphosphonate (MPn) and 1.0 mM aminoethylphosphonate in glucose-MOPS medium showed 0.5 : 1.0 ratio. In the case of BW14329 (.DELTA.phnC-P, .delta.phoA), RPI did not change significantly after 24 hrs culturing, which means it nearly could not utilize Pn. In vivo $\^$31/ P-NMR spectrum of E. aerogens (BWKL 16627) during 3 hrs starvation showed two intense peaks at 0-2 ppm and at near-10 ppm which indicate intracellular orthophosphate (Pi) and pyrophosphate (PPi), respectively. Both of them might be released by degradation of inorganic polyphosphate pool. When MPn is supplied to the medium as an unique P source, Pi content in the cell has the constant, but PPi seems to be slightly decreased. Recombinants (BWKL 16954) grew slower than E. aerogenes in the glucose-MOPS media with various P sources. In vivo $\^$31/ P-NMR spectrum of recombinant did not show any intense signal in the cell. Surprisingly, under the cultivation adding with MPn, a few intense peaks in the region of Pi AND phospate monoester were detected.

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Stable Isotope Labeling of Proteins in Mammalian Cells

  • Lee, KyungRyun;Lee, Jung Ho
    • Journal of the Korean Magnetic Resonance Society
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    • v.24 no.3
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    • pp.77-85
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    • 2020
  • Stable isotope enrichment in proteins is necessary for high-resolution nuclear magnetic resonance (NMR) experiments. Although methods for 13C, 15N and 2H-enrichment in prokaryotic cells are well established, full processing and correct folding of complex protein systems require higher organisms as the expression host. In the present study, we review recent efforts to enrich stable isotopes in mammalian cells for protein NMR studies.

Metabolic Discrimination of Rice Cultivars and Relative Quantification of Major Sugar Compounds Using 1H NMR Spectroscopy Combined by Multivariate Statistical Analysis (1H NMR 스펙트럼 데이터의 다변량 통계분석에 의한 벼 품종의 구분 및 주요 당 화합물의 정량분석)

  • Kim, Suk-Weon;Koo, Bon-Cho;Kim, Jong-Hyun;Liu, Jang-Ryol
    • Journal of Plant Biotechnology
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    • v.33 no.4
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    • pp.283-288
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    • 2006
  • Discrimination of 5 rice cultivars (Sangjubyeo , Dongjinbyeo Simbaekbyeo , Hwamanbyeo , and Simbaek-hetero ) using metabolic profiling was carried out. Whole cell extracts from each cultivar were subjected to $^1H$ NMR spectroscopy. When spectral data were analyzed by principal component analysis, 5 cultivars were clustered into 3 groups: SJ, DJ + SB, and HM + SH. Thecultivars showed great difference in carbohydrate region of $^1H$ NMR spectra, suggesting that qualitative and quantitative differences in carbohydrate compounds play a major role in discrimination of the cultivars. In addition, it was readily possible to determine relative quantification of major carbohydrates including sucrose, glucose, maltose from spectral data of the cultivars. SJ showed 2 to 4 times higher content of maltose than the other rice cultivars. Overall results indicate that metabolic discrimination of rice cultivars using $^1H$ NMR spectroscopy combined by multivariate statistical analysis can be used for rapid discrimination of numerous rice cultivars and simple quantitative analysis system of major carbohydrate compounds in rice grains.