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Medium Optimization and Application of Affinity Column Chromatography for Trypsin Production from Recombinant Streptomyces griseus

  • Chi, Won-Jae;Song, Ju-Hyun;Oh, Eun-A.;Park, Seong-Whan;Chang, Yong-Keun;Kim, Eung-Soo;Hong, Soon-Kwang
    • Journal of Microbiology and Biotechnology
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    • v.19 no.10
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    • pp.1191-1196
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    • 2009
  • The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and $50^{\circ}C$, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to $70^{\circ}C$. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

Diagnosis of Graft-Versus-Host Disease after Bone Marrow Transplantation by in vivo Proton MR Spectroscopy of the Liver: Correlation with Pathologic Results

  • Cho, Soon-Gu;Lee, Moon-Hee;Suh, Chang-Hae
    • Proceedings of the KSMRM Conference
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    • 2001.11a
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    • pp.135-135
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    • 2001
  • Purpose: To know the differences of the proton MR spectroscopic features of the liver between th patients with graft-versus-host disease (GVHD) and without GVHD (non-GVHD) after to marrow transplantation (BMT), and to evaluate the possibility to discriminate GVHD fro non-GVHD by analysis of the in vivo proton MR spectra. Method: We evaluated the in vivo proton MR spectra from the livers of 37 patients wh underwent BMT. Our series included 14 cases with GVHD and 23 without GVHD in the liver. Nineteen men and 18 women were included in our series. All cases of GVHD and 2 o non-GVHD were confirmed by liver biopsy and remaining of non-GVHD by evaluation clinical follow up. Proton MR spectroscopy (1H-MRS) was performed at 1.5T GE Sign Horizon (GE Medical System, Milwaukee, USA) system using localized proton STEAM sequence and body coil in all cases with subjects were located in supine position. N respiratory interruption was required during the spectroscopic signal acquisition. Paramete using in MRS were: TR = over 3000ms, TE = 30ms, number of scans = 128, voxel size = ($2{\times}2{\times}2$)$cm^3$, and one NEX. We evaluated the spectra with an attention to the differences o patterns of the peaks between GVHD and non-GVHD groups. The ratio of peak area of peaks at 1.6-4.1ppm to lipid (0.9-1.6ppm) [P(1.6-4.1ppm)/P(0.9-1.6ppm)] was calculated in GVHD and non-GVHD group, and compared the results between these groups. We als evaluated the sensitivity and specificity for discriminating GVHD from non-GVHD by anal of 1H-MRS.

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Toluene Induces Depression-Like Behaviors in Adult Mice

  • Yang, Mi-Young;Kim, Sung-Ho;Kim, Jong-Choon;Shin, Tae-Kyun;Moon, Chang-Jong
    • Toxicological Research
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    • v.26 no.4
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    • pp.315-320
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    • 2010
  • It has been clinically reported that toluene causes mental depression in humans. However, the detrimental effects of toluene exposure on brain function and the relation between features of mental depression and toluene exposure are poorly understood. This study evaluated depression-like behaviors in adult C57BL/6 mice after administration of toluene, and elucidated the effects of classical antidepressants on the depression-like behaviors. For the estimation of depression-like behaviors, tail suspension test (TST) and forcedswim test (FST) were performed 1, 4 and 16 days after toluene (0~1000 mg/kg bw) treatment. In addition, classical antidepressants such as fluoxetine (FLX, 20 mg/kg bw) and imipramine (IMI, 40 mg/kg bw) were administered 12 h and 1 h before the tests. In the TST and FST, toluene-treated mice exhibited a longer duration of immobility than vehicle-treated mice 1 and 4 days after toluene treatment. The depression-like behaviors were significantly reversed by FLX and IMI. The weight of the adrenal gland and the size of adrenocortical cells were significantly higher in toluene-treated mice compared to vehicle-treated controls. It is suggested that acute toluene exposure of adult mice is sufficiently detrimental to induce depression. In addition, this study has established a mouse model for a depressive state induced by toluene treatment.

Evaluation of Haemagglutinin Content by RP-HPLC to Generate Pandemic Influenza Vaccine

  • Kang, Hyunkyung;Roh, Hang Sik;Song, Hyemin;Lee, Kwangmoon;Chung, Seung-Tae;Ban, Sang-ja;Mo, In Pil;An, Beum-Soo;Ahn, Chi-Young
    • Toxicological Research
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    • v.32 no.4
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    • pp.269-274
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    • 2016
  • The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.

Study on the Simple Preparation Method of Honeycomb-structured Catalysts by Temperature-regulated Chemical Vapor Deposition (온도조절 화학기상증착법을 활용한 대용량 허니컴 구조촉매 제조 연구)

  • Seo, Minhye;Kim, Soong Yeon;Kim, Young Dok;Uhm, Sunghyun
    • Applied Chemistry for Engineering
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    • v.29 no.1
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    • pp.18-21
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    • 2018
  • We report on the simple preparation method of large-scale structured catalysts by temperature-regulated chemical vapor deposition with a high cell-density ceramic honeycomb monolith. And the feasibility for dry reforming of methane catalysts was evaluated. The NiO/Cordierite (CDR) catalyst was prepared by controlling coating conditions at each temperature step, leading to a conformal deposition of NiO inside the cordierite honeycomb monolith with the cell density of 600 cpsi. The catalytic conversion of $CH_4$ and $CO_2$ for dry reforming of methane were about 83% and 90% with gas hourly space velocity of $10,000h^{-1}$ at $800^{\circ}C$, respectively. As a result, it exhibited that the temperature-regulated chemical vapor deposition method can be expedient for the preparation of large-scale structured catalysts.

Comparison of Core Muscle Activity and Thickness According to Walking Training Method (워킹 훈련방법에 따른 복부 중심근육 활성도와 근 두께 변화 비교)

  • Lee, H.J.;Kim, Y.T.;Lee, S.J.;Kim, M.S.;Kim, S.H.;Tae, K.S.
    • Journal of rehabilitation welfare engineering & assistive technology
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    • v.9 no.4
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    • pp.301-308
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    • 2015
  • The purpose of this study was to compare core muscle activity and thickness in the abdomen (internal Oblique, IO; External Oblique, EO; Transverse Abdominis, TrA) according to walking training methods. Tests were performed on 20 healthy men who randomly assigned to two groups, divided by Nordic walking (n=10) or Power walking group (n=10). They were performed Nordic walking or Power walking training for 2 weeks that is consistent with each of the assigned groups. Results demonstrated that Nordic walking was more effective than Power walking in improving IO and EO activities. Nordic walking is believed to be useful method for a variety of therapeutic exercise as a stable balance with the stick in addition to normal gait and trunk stability.

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Endovaginal and Endorectal Surface Coils for in-ViVo Human MR Imaging and Spectroscopy (자궁경부암 진단용 MR 질내표면코일과 전립선암 진단용 MR 경직장표면코일의 제작 : 인체에서의 MR 영상과 MR 분광)

  • 문치웅;조경식
    • Journal of Biomedical Engineering Research
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    • v.16 no.4
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    • pp.481-491
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    • 1995
  • Endovaginal and endorectal receiver only surface coils were designed for MR imaging (MRI) and $^1H$ MR spectroscopy (MRS) for the uterine cervix and the prostate. The shape of endovaginal coil wire was rectangular with round corner. Size of the coil wire was empirically determined for 7cm and 4cm along the long and short axis, respectively. The coil wire loop was supported by acryl handle and bent about $150^{\circ}$ at one side of the loop considering the average angle of the cervix to the vagina. We called this as a "spoon-type endovaginal coil". The wire of the endorectal coil was made of the flexible materials so that the wire loop became long elliptic shape by pushing the acryl handle into the plastic tube for the comfort of patients when the coil was inserted into the cervix. Then, the shape was maintained to be circle by popping out handle. Conventional spin echo (SE) and fast spin echo (FSE) sequences were used as 71 and 72 weighted imaging sequences, respectively. Matrix size was 128~$256{\times}256$. FOVs for surface coil and body coil were 14cm and 24cm, respectively. 3D volume localized in vivo $^1H$ MR spectroscopy of the human cervix and prostate was performed using PRESS or STEAM localization method with the following parameters . TR=3 sec, TE=135 msec for PRESS or 30 msec for STEAM, NEX=2, NS=48, Sl=2048, and SW=2500 Hz. Using home-built endovaginal and endorectal coils, excellent T1- and T2-images were obtained to visualize early cervical and prostate tumors. 3D volume localized in vivo IH MRS was useful to differentiate the cancerous tissue from the normal tissue.

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Identification of Genes Connected with the Sensitivity to 5-FU and Cisplatin in Squamous Cell Carcinoma Cell Lines (편평세포암 세포주에서 5-FU와 Cisplatin에의 감수성과 관련된 유전자의 동정)

  • Choi, Na-Young;Kim, Ok-Joon;Lee, Geum-Sug;Kim, Byung-Gook;Kim, Jae-Hyeong;Jang, Youn-Young;Lim, Won-Bong;Chong, Min-A;Choi, Hong-Ran
    • Journal of Oral Medicine and Pain
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    • v.30 no.3
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    • pp.287-300
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    • 2005
  • Squamous cell carcinoma (SCC) in head and neck show a variability in the response to chemotherapy, even when it present with similar histological tumor type, grade, and clinical stage. The purpose of present study it to identify predictive bio-marker for the sensitivity or resistance to conventional chemotherapeutic agents, 5-fluorouracil (5-FU) and Cisplatin Oral cancer cell lines were used in present study. MTT assay was performed to evaluate the sensitivity and/or resistance to 5-FU and Cisplatin. And RT-PCR was carried out for evaluation of the mRNA expressions of various genes associated with mutation, inflammation (COX pathway), cell cycle, senescence and extracellular matrix (ECM). The molecules which are correlated with the sensitivity to 5-FU are XPA, XPC, OGG, APEX, COX-2, PPAR, Cyclin E, Cyclin B1, CDC2, hTERT, hTR, TIMP-3, TIMP-4 and HSP47. And the molecules are correlated with the sensitivity to Cisplatin are COX-1, iNOS, eNOS, PCNA, collagen 1 and MMP-9. Taken together, when choosing the appropriate chemotherpeutic agents for patients, considering the molecules which are correlated or reversely correlated is helpful to choose the resonable agents for cancer patients.

Effects of Water-misting Sprays with Forced Ventilation after Transport during Summer on Meat Quality, Stress Parameters, Glycolytic Potential and Microstructures of Muscle in Broilers

  • Jiang, N.N.;Xing, T.;Wang, P.;Xie, C.;Xu, X.L.
    • Asian-Australasian Journal of Animal Sciences
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    • v.28 no.12
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    • pp.1767-1773
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    • 2015
  • Effects of water-misting sprays with forced ventilation after transport during summer on meat quality, stress parameters, glycolytic potential and microstructures of muscle in broilers were investigated. A total of 105 mixed-sex Arbor Acres broilers were divided into three treatment groups: i) 45-min transport without rest (T group), ii) 45-min transport with 1-h rest (TR group), iii) 45-min transport with 15-min water-misting sprays with forced ventilation and 45-min rest (TWFR group). The results showed the TWFR group significantly increased (p<0.05) initial muscle pH ($pH_i$) and ultimate pH ($pH_u$) and significantly reduced $L^*$ (p<0.05), drip loss, cook loss, creatine kinase, lactate dehydrogenase activity, plasma glucose content, lactate and glycolytic potential when compared with other groups. Microstructure of the muscle from TWFR group broilers under light microscopy showed smaller intercellular spaces among muscle fibers and bundles compared with T group. In conclusion this study indicated water-misting sprays with forced ventilation after transport could relieve the stress caused by transport under high temperature, which was favorable for the broilers' welfare. Furthermore, water-misting sprays with forced ventilation after transport slowed down the postmortem glycolysis rate and inhibited the occurrence of PSE-like meat in broilers. Although rest after transport could also improve the meat quality, the effect was not as significant as water-misting sprays with forced ventilation after transport.

[ $^1H$ ] MR Spectroscopy of the Normal Human Brains: Comparison between Signa and Echospeed 1.5 T System (정상 뇌의 수소 자기공명분광 소견: 1.5 T Signa와 Echospeed 자기공명영상기기에서의 비교)

  • Kang Young Hye;Lee Yoon Mi;Park Sun Won;Suh Chang Hae;Lim Myung Kwan
    • Investigative Magnetic Resonance Imaging
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    • v.8 no.2
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    • pp.79-85
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    • 2004
  • Purpose : To evaluate the usefulness and reproducibility of $^1H$ MRS in different 1.5 T MR machines with different coils to compare the SNR, scan time and the spectral patterns in different brain regions in normal volunteers. Materials and Methods : Localized $^1H$ MR spectroscopy ($^1H$ MRS) was performed in a total of 10 normal volunteers (age; 20-45 years) with spectral parameters adjusted by the autoprescan routine (PROBE package). In all volunteers, MRS was performed in a three times using conventional MRS (Signa Horizon) with 1 channel coil and upgraded MRS (Echospeed plus with EXCITE) with both 1 channel and 8 channel coil. Using these three different machines and coils, SNRs of the spectra in both phantom and volunteers and (pre)scan time of MRS were compared. Two regions of the human brain (basal ganglia and deep white matter) were examined and relative metabolite ratios (NAA/Cr, Cho/Cr, and mI/Cr ratios) were measured in all volunteers. For all spectra, a STEAM localization sequence with three-pulse CHESS $H_2O$ suppression was used, with the following acquisition parameters: TR=3.0/2.0 sec, TE=30 msec, TM=13.7 msec, SW=2500 Hz, SI=2048 pts, AVG : 64/128, and NEX=2/8 (Signa/Echospeed). Results : The SNR was about over $30\%$ higher in Echospeed machine and time for prescan and scan was almost same in different machines and coils. Reliable spectra were obtained on both MRS systems and there were no significant differences in spectral patterns and relative metabolite ratios in two brain regions (p>0.05). Conclusion : Both conventional and new MRI systems are highly reliable and reproducible for $^1H$ MR spectroscopic examinations in human brains and there are no significant differences in applications for $^1H$ MRS between two different MRI systems.

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