• Title/Summary/Keyword: $^{15}N$ labeling

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Dynamics of the mobile insert helix in the domain III-IV of Aux/IAA17 probed by site-directed spin labeling and paramagnetic NMR spectroscopy

  • Han, Mookyoung;Suh, Jeong-Yong
    • Journal of the Korean Magnetic Resonance Society
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    • v.19 no.2
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    • pp.61-66
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    • 2015
  • The plant hormone auxin is involved in all stages of plant development. Aux/IAAs are the transcriptional repressors that bind to the Auxin Response Factors (ARFs) to regulate the gene expression upon auxin release. Aux/IAA have highly conserved C-terminal domains (domains III-IV) that mediate both homotypic and heterotypic interactions between Aux/IAA and ARF family proteins. Recent studies revealed that the conserved domains III-IV share a common ${\beta}$-grasp fold that oligomerizes in a front-to-back manner. In particular, Aux/IAA contains a mobile insert helix in the domain III-IV, whereas ARFs do not. Here, we investigated the dynamics of the insert helix using paramagnetic NMR spectroscopy. The insert helix exhibited fast motions in the ps-ns time scale from $^{15}N$ relaxation data, but the amplitude of the motion is likely limited to the local neighborhood. Our result suggests that the motion of the helix may have functional implications in protein-protein interactions for transcriptional regulations.

Paratope Mapping of Anti-Ex-A IgG as Studied by NMR (NMR에 의한 anti-Ex-A IgG의 항원결합부위 해석)

  • Kim, Ha-Hyeong;Lee, Gwang-Pyo
    • YAKHAK HOEJI
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    • v.40 no.4
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    • pp.422-427
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    • 1996
  • The anti-Ex-A IgG was specifically labeled with stable isotopes, DL-His-2,4-$d_2$, L-Phe-$d_5$, L-Trp-$d_5$, L-Tyr-2,6-$d_2$ and L-[1-$^{13}C$]Trp, by growing hybridoma cell in serum-free medium. By use of NMR spectroscopy with selectively labeled Fab fragment, we applied a paratope mapping on antigen-antibody complex. Assignments of the observed carbonyl carbon resonances have been determined by using $^{13}C$-$^{15}N$ double labeling method in order to assign the Trp resonances. Photo CIDNP was also applied to investigate the antigen-binding site(s) on the surface residues of antibody. We found that Trp 36, which is located at the $V_H$ domain, is an important residue to bind to Ex-A, however, two Tyr on the surface of anti-Ex-A IgG plays no crucial role to bind to antigen. On the basis of these results, we demonstrate that stable isotope-aided NMR strategy can be extended to molecular structural analyses of the complex of an Fab fragment and a protein antigen.

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NMR study of the interaction of T4 Endonuclease V with DNA

  • Lee, Bong-Jin;Im, Hoo-Kang;Hyungmi Lihm;Yu, Jun-Suk
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1995.04a
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    • pp.80-80
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    • 1995
  • T4 Endonuclease V (Mw 16,000) acts as a repair enzyme for UV induced pyrimidine dimers in DNA. Many researchers have studied the biochemical characteristics of the enzyme. However the precise action mechanism of T4 endo V has not fully elucidated yet. In our laboratory NMR spectroscopy technique is being used for the structural study of T4 endo V. Because of its low temperature stability and high content of ${\alpha}$-helix, the conventional $^1$H NMR technique was inapplicable. Therefore we utilized stable isotope labeling technique and so far prepared about 10 amino acid specific labeled proteins. The HSQC spectra of amino acid specific labeled proteins will help us to interpret the triple resonance 3D, 4D data which are under processing, We also studied the behaviors of specific amino acid residues whose roles might be critical. When the enzyme labeled by $\^$15/N-Thr was mixed with the substrate oligonucleotide (semispecific -TT- sequence), one crosspeak in its HSQC spectrum was completely desappeared, which means that one of seven Thr residues is in the binding site of the enzyme with DNA, This result is well consistent with previous report that implicated the Thr 2 residue in the activity of the enzyme. Similar studies were carried on the behaviors of Arg and Tyr residues.

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Recombinant Expression, Isotope Labeling, and Purification of Cold shock Protein from Colwellia psychrerythraea for NMR Study

  • Moon, Chang-Hun;Jeong, Ki-Woong;Kim, Hak-Jun;Heo, Yong-Seok;Kim, Yang-Mee
    • Bulletin of the Korean Chemical Society
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    • v.30 no.11
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    • pp.2647-2650
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    • 2009
  • Cold shock proteins (Csps) are a subgroup of the cold-induced proteins on reduction of the growth temperature below the physiological temperature. They preferentially bind to single-stranded nucleic acids to translational regulation via RNA chaperoning. Csp plays important role in cold adaptations for the psychrophilic microorganism. Recently, Cold shock protein from psychrophilic bacteria, Colwellia psychrerythraea (CpCsp) has been identified. Three dimensional structures of a number of Csps from various microorganisms have been solved by NMR spectroscopy or X-ray crystallography, but structures of psychrophilic Csps were not studied yet. Therefore, cloning and purification protocols for further structural study of psychrophilic Csp have been optimized in this study. CpCsp was expressed in E. coli with pET-11a vector system and purified by ion exchange, size exclusion, and reverse phase chromatography. Expression and purification of CpCsp in M9 minimal media was carried out and $^{15}N$-labeled proteins with high purity over 90% was obtained. Further study will be carried out to investigate the tertiary structure and dynamics of CpCsp.

Evaluation of 99mTc-MAG3-2-nitroimidazole for hypoxic tumor imaging

  • Lee, Yun-Sang;Kim, Young Joo;Jeong, Jae Min
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.5 no.1
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    • pp.18-25
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    • 2019
  • 2-Nitroimidazole derivatives have been reported to accumulate in hypoxic tissue. We prepared a novel $^{99m}Tc-MAG_3$-2-nitroimidazole and evaluated the feasibility for hypoxia imaging agent. $Bz-MAG_3$-2-nitroimidazole was synthesized by direct coupling of $Bz-MAG_3$ and 2-nitroimidazole using dicyclohexylcarbodiimide. $Bz-MAG_3$-2-nitroimidazole was labeled with $^{99m}Tc$ in the presence of tartaric acid and $SnCl_2-2H_2O$ at $100^{\circ}C$ for 30 min. And the reaction mixture was purified by $C_{18}$ Sep-pak cartridge. The labeling efficiency and the radiochemical purity were checked by ITLC-SG/acetonitrile. The tumor was grown in balb/c mice for 8~13 days after the subcutaneous injection of tumor cells, CT-26 (murine colon adenocarcinoma cell). Biodistribution study and tumor autoradiography were performed in the xenografted mice after i.v injection of 74 kBq/0.1 mL and 19 MBq/0.1 mL of $^{99m}Tc-MAG_3$-2-nitroimidazole, respectively. In vivo images of $^{99m}Tc-MAG_3$-2-nitroimidazole in tumor bearing mice were obtained 1.5 hr post injection. The labeling efficiency was $45{\pm}20%$ and the radiochemical purity after purification was over 95%. Paper electrophoresis confirmed negative charge of $^{99m}Tc-MAG_3$-2-nitroimidazole. $^{99m}Tc-MAG_3$-2-nitroimidazole was very stable at room temperature and its protein binding was 53%. The $^{99m}Tc-MAG_3$-2-nitroimidazole exhibited high uptake in the liver, stomach and intestine. In biodistribution study using tumor bearing mice, the uptakes (% ID/g) of the tumor were $0.5{\pm}0.1$, $0.4{\pm}0.0$, $0.2{\pm}0.1$ and $0.1{\pm}0.1$ at 5, 15, 30 min and 4 hrs. Tumor/muscle ratio were $1.4{\pm}0.1$, $2.2{\pm}0.83$, $3.0{\pm}0.9$, and 3.7 (n=2) for 5, 15, 30 min and 4 hrs. The uptake in hypoxic area was found higher than in non-hypoxic area of tumor tissue by autoradiography. In vivo images showed the relatively faint uptake to the hypoxic tumor region. $^{99m}Tc-MAG_3$-2-nitroimidazole was successfully synthesized and found feasible for imaging hypoxia.

Nitrogen Partitioning at Low Temperature in Fall-Sowing Species II. Distribution to roots, xylem and phloem transport of newly absorbed nitrate (추파 청예작물의 저온 조건하에서 질소의 분배에 관한 연구 II. 흡수된 질산태 질소의 목부, 체관부 및 뿌리로의 전이)

  • Kim, T.H.;Kim, B.H.
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.19 no.1
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    • pp.49-56
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    • 1999
  • With ${15}^N$ labeling under split roots system of winter rye (Secale cereale L.) and forage rape (Brassica napus L.) grown at $5^{\circ}C$ and $25^{\circ}C$, the N flows were respectively quantified to investigate the transport of newly absorbed nitrate-N in whole plant level at low temperature. Comparing with $25^{\circ}C$ culture condition, the total absorbed nitrate-N content at $5^{\circ}C$ decreased to 59.3% and 27.1% in winter rye and forage rape during 9 days. About 2.5% and 7.6 % of nitrate-N were transported into roots, respectively, in winter rye and in forage rape at $25^{\circ}C$. These proportions increased at $5^{\circ}C$ to 3.8% and 10.9%, respectively. Total N contents transferred by xylem in winter rye grown and forage rape grown at $25^{\circ}C$ during were 55.9 and 54.4 mg N/plant, respectively. xylem flows at $5^{\circ}C$ were 60.4% and 28.8% lower than at $25^{\circ}C$ for winter rye and forage rape. These valves represented that averagely 96.8 % and 90.8% of total absorbed nitrate-N were transferred to leaves in winter rye and forage rape during 9 days. Phloem flows were the smallest among other N flows and were much less influenced by temperature treatment for two species examined. About 2.5% and 0.5% of absorbed N were recycled into roots by phloem transport at $25^{\circ}C$, respectively, for winter rye and forage rape. These proportions increased to 5.2% and 0.9% at $5^{\circ}C$.

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Expression of Neuronal Nitric Oxide Synthase (nNOS) in Developing Rat Kidney (분화중인 흰쥐 콩팥의 요세관에서 nNOS의 발현)

  • Song, Ji-Hyun;Ryu, Si-Yun;Kim, Jin;Jung, Ju-Young
    • Applied Microscopy
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    • v.38 no.2
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    • pp.141-148
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    • 2008
  • Nitric oxide (NO) is an important regulator of renal blood flow, glomerular hemodynamics, and tubule transport processes in the kidney. There is also evidence that NO is involved in cell cycle regulation and mitotic division. During development the nNOS expression pattern differs from that observed in adult animals. However, little is known about temporal and spatial patterns of nNOS expression in the developing kidney. The purpose of this study was to establish the time of expression and the distribution of nNOS in the developing rat kidney. Kidneys from 14-, 16-, 17-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were preserved and processed for immunohistochemistry. In the adult kidney, nNOS was detected in the parietal epithelium of Bowman s capsule, macula densa, descending thin limb and inner medullary collecting duct. nNOS immunoreactivity appeared first in the distal tubule anlage at 15 days of gestation, and in all epithelial cells of developing thick ascending limbs (TAL) as well as macula densa of 17- and 18-day-old fetuses. From 20 days of gestation to 14 days after birth, nNOS was expressed in the newly formed cortical TAL, which are located in the medullary ray, whereas in mature TAL of juxtamedullary nephrons, nNOS immunolabeling gradually decreased in intensity and became restricted to the macula densa. In inner medullary collecting ducts, nNOS immunoreactivity appeared first at 7 days after birth in the papillary tip and gradually ascended to the border between outer and inner medulla. In the descending thin limb and parietal epithelium of Bowman's capsule, weak nNOS immunoreactivity was observed at 14 days after birth and labeling gradually increased to adult levels at 21 days after birth. These results suggest that differential expression of nNOS in the developing kidney is an important physiological regulator of renal function during kidney maturation.

An implementation of 2D/3D Complex Optical System and its Algorithm for High Speed, Precision Solder Paste Vision Inspection (솔더 페이스트의 고속, 고정밀 검사를 위한 이차원/삼차원 복합 광학계 및 알고리즘 구현)

  • 조상현;최흥문
    • Journal of the Institute of Electronics Engineers of Korea SP
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    • v.41 no.3
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    • pp.139-146
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    • 2004
  • A 2D/3D complex optical system and its vision inspection algerian is proposed and implemented as a single probe system for high speed, precise vision inspection of the solder pastes. One pass un length labeling algorithm is proposed instead of the conventional two pass labeling algorithm for fast extraction of the 2D shape of the solder paste image from the recent line-scan camera as well as the conventional area-scan camera, and the optical probe path generation is also proposed for the efficient 2D/3D inspection. The Moire interferometry-based phase shift algerian and its optical system implementation is introduced, instead of the conventional laser slit-beam method, for the high precision 3D vision inspection. All of the time-critical algorithms are MMX SIMD parallel-coded for further speedup. The proposed system is implemented for simultaneous 2D/3D inspection of 10mm${\times}$10mm FOV with resolutions of 10 ${\mu}{\textrm}{m}$ for both x, y axis and 1 ${\mu}{\textrm}{m}$ for z axis. Experiments conducted on several nBs show that the 2D/3D inspection of an FOV, excluding an image capturing, results in high speed of about 0.011sec/0.01sec, respectively, after image capturing, with $\pm$1${\mu}{\textrm}{m}$ height accuracy.

Social Interaction of Caregivers and Their Children with Down Syndrome or Without Disability (다운증후군 아동과 정상아동의 보호자와의 사회적 상호관계에 대한 비교연구)

  • Cho, Mi-hyun;Cho, mi-suk
    • Proceedings of the Korea Contents Association Conference
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    • 2009.05a
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    • pp.1076-1082
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    • 2009
  • This study investigated caregivers' communication styles and children's emotional development. Emotion-laden puzzle tasks were used to elicit caregivers' communication styles while interacting with their children. Participants included children with Down syndrome (N=10) and typical children (N=15) and their caregivers. As expected, caregivers of children with Down syndrome (DS) used more behavior and attention directives with their children, and caregivers of typical children used more conversation-eliciting prompts with their children. Parents of children with Down syndrome also used a unique communication style in which they asked a question and immediately answered it themselves. Additionally, caregivers of typical children focused more on emotion concepts in their communications with their children and caregivers of DS used more cognitive concepts such as labeling colors and shapes. The results revealed that caregivers of children with Down syndrome usually tried to educate children by emphasizing cognitive concepts to compensate for their delayed development. Because the children are delayed in their emotional development, parents may need help in intervening on the area of emotional development.

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Energy and nutrition evaluation per single serving package for each type of home meal replacement rice (가정간편식 밥류의 유형별 1회 제공 포장량 당 에너지 및 영양성분 함량 평가)

  • Choi, In-Young;Yeon, Jee-Young;Kim, Mi-Hyun
    • Journal of Nutrition and Health
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    • v.55 no.4
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    • pp.476-491
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    • 2022
  • Purpose: The purpose of this study was to evaluate the energy and nutrient contents of home meal replacement (HMR) rice products per single serving package based on nutrition labels. Methods: The market research was conducted from February to July 2021 on products sold on the internet, at convenience stores, etc. A total of 406 products were investigated. The products were divided into the following 6 classifications: instant rice (n = 45), cup rice (n = 64), frozen rice (n = 188), rice bowls with toppings (n = 32), gimbap (n = 38), and triangular gimbap (n = 39). Results: The mean packaging weight per serving was the highest in the rice bowl with toppings at 297.1 g, followed by cup rice (264.0 g), frozen rice (239.5 g), gimbap (230.2 g), instant rice (193.4 g), and triangular gimbap (121.6 g) (p < 0.001). The energy per serving package for the rice bowl with toppings was significantly the highest at 496.0 kcal (p < 0.001). The sodium content per serving package of gimbap was the highest at 1,021.8 mg and that of the instant rice was lowest at 37.4 mg (p < 0.001). The price per serving package of the rice bowl with toppings at 4,333.8 won was the highest. The contribution to the daily nutritional value per serving package of all types of HMR rice products surveyed showed an average range of 10-25% for energy, 11-22% for carbohydrates, and 2-51% for sodium. Conclusion: These results indicate the energy and nutrient contents of HMR rice products, vary by type. Therefore, consumers should review the nutrition labeling to select an appropriate HMR rice product based on their intended consumption.