• Journal of Life Science
• /
• v.19 no.8
• /
• pp.1039-1046
• /
• 2009

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

• Journal of Life Science
• /
• v.24 no.8
• /
• pp.851-859
• /
• 2014

The present study was designed to investigate whether ethanol extracts of Sophora flavescens (GS), Glycyrrhiza uralensis (GC), Dictamnus dasycarpus (BSP), and their mixtures (GGB-1, -2, -3, and -4) inhibit 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD) in a mouse model. DNCB was topically applied on the dorsal surface of Balb/c mice to induce AD-like skin lesions. The pathological phenotypes of AD, such as erythema, ear thickness, edema, scabs, and discharge, were significantly decreased in the GGB (DNCB + GS:GC:BSP = 3:1:1 mixture)-1-treated groups compared with the other treated groups. The weight of the spleen in immune organs was significantly decreased in the GGB-1-treated groups, whereas the weight of the liver in a control group was similar to that of the groups treated with the samples. Furthermore, toluidine blue staining analysis, a method used to specifically identify mast cells, showed that master cell infiltration into the dermis of the GGB-1-treated group was significantly decreased. The immunoglobulin E concentration was lower in the GGB-1-treated group. In addition, the levels of inflammatory cytokines (interferon-${\gamma}$, interleukin-1, 4, 5, 6, and 13, $1{\beta}$, and tumor necrosis factor-${\alpha}$) were also significantly reduced in the GGB-1-treated group. Taken together, these results suggest that a mixture of GS, GC, and BSP in a proportion of 3:1:1 (GGB-1) may contribute to the relief of AD symptoms and may be considered an excellent candidate for an AD therapeutic drug.

• Journal of Life Science
• /
• v.28 no.10
• /
• pp.1220-1232
• /
• 2018

The purpose of this animal study was to evaluate, by histological analysis, bone regeneration in rabbit maxillary sinuses with an anorganic bovine graft (Bio-Oss) and a ${\beta}-tricalcium$ phosphate (${\beta}-TCP$) grafting. Bilateral sinus augmentation procedures were performed in 12 adult male rabbits. Rectangular replaceable bony windows were made with a piezoelectric thin saw insert. In the Bio-Oss group, Bio-Oss was grafted and in the ${\beta}-TCP$ group, ${\beta}-TCP$ was grafted and covered by replaceable bony windows. The animals were sacrificed at 2, 4, and 8 weeks after the surgical procedure. The augmented sinuses were evaluated by histomorphometric analysis using hematoxylin-eosin, Masson trichrome, and tartrate-resistant acid phosphatase stains and also by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), type I collagen, and osteocalcin content. Histologically, new bone formation was found on the surface of Bio-Oss and ${\beta}-TCP$ particles from 2 weeks and continued to 8 weeks. Significant higher new bone formation was revealed in the ${\beta}-TCP$ group than in the Bio-Oss group at 8 weeks. The amount of graft materials was significantly decreased in the ${\beta}-TCP$ group and the number of osteoclasts was significantly increased in the ${\beta}-TCP$ group from 4 to 8 weeks. Immunoreactivity to PCNA was reduced at 8 weeks. The expression of type I collagen was significantly increased in the ${\beta}-TCP$ group at 2 weeks, but was significantly increased in the Bio-Oss group at 8 weeks. Immunoreactivity to osteocalcin was increased from 2 to 8 weeks. These histological results can help in the selection of graft materials for implants. Both Bio-Oss and ${\beta}-TCP$ are proven graft materials, however, these results indicate that ${\beta}-TCP$ showed better bone regeneration results in rabbit maxillary sinus augmentation.

• Journal of Life Science
• /
• v.30 no.11
• /
• pp.947-955
• /
• 2020

The foot-and-mouth disease virus (FMDV), a member of the Aphthovirus genus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. During replication of the FMDV RNA (ribonucleic acid) genome, FMDV-encoding RNA polymerase 3D acts in a highly location-specific manner. This suggests that specific RNA structures recognized by 3D polymerase within non-coding regions of the FMDV genome assist with binding during replication. One such region is the cis-acting replication element (CRE), which functions as a template for RNA replication. The FMDV CRE adopts a stem-loop conformation with an extended duplex stem, supporting a novel 15-17 nucleotide loop that derives stability from base-stacking interactions, with the exact RNA nucleotide sequence of the CRE producing different RNA secondary structures. Here, we show that CRE sequences of FMDVs isolated in Korea from 2010 to 2017 exhibit A and O genotypes. Interestingly, variations in the RNA secondary structure of the Korean FMDVs are consistent with the phylogenetic relationships between these viruses and reveal the specificity of FMDV infections for particular host species. Therefore, we conclude that each genetic clade of Korean FMDV is characterized by a unique functional CRE and that the evolutionary success of new genetic lineages may be associated with the invention of a novel CRE motif. Therefore, we propose that the specific RNA structure of a CRE is an additional criterion for FMDV classification dependent on the host species. These findings will help correctly analyze CRE sequences and indicate the specificity of host species for future FMDV epidemics.

• Journal of Genetic Medicine
• /
• v.8 no.1
• /
• pp.1-16
• /
• 2011

Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

• Radiation Oncology Journal
• /
• v.18 no.4
• /
• pp.226-232
• /
• 2000

Purpose :This study was performed to find out the prognostic factors affecting local control in early glottic cancer treated with radiation therapy alone. Materials and Methods :We analysed 37 patients of histologically confirmed early glottic cancer treated at Chonnam National University Hospital between July Im and December 1995, retrospectively. Age of patients ranged from 30 to 73 years (median; 59 years). Thirty-five (95$\%$) patients were male. Histological type was all squamous cell carcinoma. According to the staging system of 1997 American Joint Committee on Cancer, 37 patients were restaged as follows: Tla; U (73$\%$), Tlb; 3 (8$\%$), 72: 7 (19$\%$). Radiation therapy was done using 6 MV X-ray of linear accelerator The range of total radiation dose delivered to the glottic lesion was between 5,040 cGy and 7,020 cGy (median; 6,600 cGy). Median follow-up period was U months. local control rates were calculated by Kaplan-Meier method. Generalized Wilcoxon test was used to evaluate the difference of control rates between comparable groups. Multivariate analysis using Cox proportional hazard model was done to find out prognostic factors affecting local control. Results:5 year survival rate of 37 patients was 89$\%$. Local control rate of 37 patients was 74$\%$ in 5 years. We included age, 7-stage, anterior commissure involvement, fraction size, total radiation dose, treatment time of radiotherapy as potential prognostic factors in univariate and multivariate analysis. As a result, treatment time had statistical significance in local control rate in both univariate (p=0.026) and multivariate (p=0.017) analysis. Complication was not recorded except one patient with hypothyroidism. Conclusion :This study revealed that overall treatment time of radiation was a significant factor affecting local control rate.

• Applied Biological Chemistry
• /
• v.50 no.2
• /
• pp.95-100
• /
• 2007

The cellulase gene of Bacillus licheniformis K11 which has plant growth-promoting activity by auxin and antagonistic ability by siderophore was cloned in pUC18 using PCR employing heterologous primers. The 1.6kb PCR fragment contained the full sequence of the cellulase gene, denoted celW which has been reported to encode a 499 amino acid protein. Similarity search in protein data base revealed that the cellulase from B. licheniformis K11 was more than 97% identical in amino acid sequence to those of various Bacillus spp. The cellulase protein from B. licheniformis K11, overproduced in E. coli DH5${\alpha}$ by the lac promoter on the vector, had apparent molecular weight of 55 kDa upon CMC-SDS-PAGE analysis. The protein not only had enzymatic activity toward carboxymethyl-cellulose (CMC), but also was able to degrade insoluble cellulose, such as Avicel and filter paper (Whatman$^{\circledR}$ No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. Consequently B. licheniformis K11 was able to suppress the peperblight causing P. capsici by its cellulase. Biochemical analysis showed that the enzyme had a maximum activity at 60$^{\circ}C$ and pH 6.0. Also, the enzyme activity was activated by Co$^{2+}$ of Mn$^{2+}$ but inhibited by Fe$^{3+}$ or Hg$^{2+}$. Moreover, enzyme activity was not inhibited by SDS or sodium azide.

• The Journal of Korean Society for Radiation Therapy
• /
• v.26 no.1
• /
• pp.59-67
• /
• 2014

Purpose : This study aims to evaluate 3D dosimetric impact for MIP image and each phase image in stereotactic body radiotherapy (SBRT) for lung cancer using volumetric modulated arc therapy (VMAT). Materials and Methods : For each of 5 patients with non-small-cell pulmonary tumors, a respiration-correlated four-dimensional computed tomography (4DCT) study was performed. We obtain ten 3D CT images corresponding to phases of a breathing cycle. Treatment plans were generated using MIP CT image and each phases 3D CT. We performed the dose verification of the TPS with use of the Ion chamber and COMPASS. The dose distribution that were 3D reconstructed using MIP CT image compared with dose distribution on the corresponding phase of the 4D CT data. Results : Gamma evaluation was performed to evaluate the accuracy of dose delivery for MIP CT data and 4D CT data of 5 patients. The average percentage of points passing the gamma criteria of 2 mm/2% about 99%. The average Homogeneity Index difference between MIP and each 3D data of patient dose was 0.03~0.04. The average difference between PTV maximum dose was 3.30 cGy, The average different Spinal Coad dose was 3.30 cGy, The average of difference with $V_{20}$, $V_{10}$, $V_5$ of Lung was -0.04%~2.32%. The average Homogeneity Index difference between MIP and each phase 3d data of all patient was -0.03~0.03. The average PTV maximum dose difference was minimum for 10% phase and maximum for 70% phase. The average Spain cord maximum dose difference was minimum for 0% phase and maximum for 50% phase. The average difference of $V_{20}$, $V_{10}$, $V_5$ of Lung show bo certain trend. Conclusion : There is no tendency of dose difference between MIP with 3D CT data of each phase. But there are appreciable difference for specific phase. It is need to study about patient group which has similar tumor location and breathing motion. Then we compare with dose distribution for each phase 3D image data or MIP image data. we will determine appropriate image data for treatment plan.

• Restorative Dentistry and Endodontics
• /
• v.32 no.6
• /
• pp.498-513
• /
• 2007

Since it was reported that incipient enamel caries can be recovered, previous studies have quantitatively evaluated that enamel artificial caries have been, remineralized with fluoride showing simultaneously the increase of width of surface layer and the decrease of width of the body of legion. There is, however, little report which showed that remineralization could occur without fluoride. In addition, the observations on the change of hydroxyapatite crystals also have been scarcely seen. In this study, enamel caries in intact premolars or molars was induced by using lactic acidulated buffering solutions over 2 days. Then decalcified specimens were remineralized by seven groups of solutions using different degree of saturation(0.212, 0.239, 0.301, 0.355) and different pH(5.0, 5.5, 6.0) over 10 days. A qualitative comparison to changes of hydroxyapatite crystals after fracturing teeth was made under SEM(scanning electron microscopy) and AFM(atomic force microscopy). The results were as follows: 1. The size of hydroxyapatite crystals in demineralized area was smaller than the normal ones. While the space among crystals was expanded, it was observed that crystals are arranged irregularly. 2. In remineralized enamel area, the enlarged crystals with various shape were observed when the crystals were fused and new small crystals in intercrystalline spaces were deposited. 3. Group 3 and 4 with higher degree of saturation at same pH showed the formation of large clusters by aggregation of small crystals from the surface layer to the lesion body than group 1 and 2 with relatively low degree of saturation at same pH did. Especially group 4 showed complete remineralization to the body of lesions. Group 5 and 6 with lower pH at similar degree of saturation showed remineralization to the body of lesions while group 7 didn't show it. Unlike in Group 3 and 4, Group 5 and 6 showed that each particle was densely distributed with clear appearance rather than crystals form clusters together.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.32 no.2
• /
• pp.197-206
• /
• 2003

Physiological functions of raw grain diet composed of brown rice and Job's Tear (1 : 1) were evaluated in rats raised with nutritionally unbalanced diet including 1% cholesterol, high proportion of animal lipids (lard: soybean oil : 8 : 2), sub-optimal levels of vitamin and mineral mixture along with 0.5% ethanol in drinking water for 4 weeks. Control rats were fed the AIN-93G diet for 9 weeks, and nutritionally unbalanced rats were divided into 3 groups, and fed one of the following diets with 0.5% ethanol in drinking water for another 5 weeks: unbalanced control diet (UCD), raw grain diet (RGD) (UCD +20% brown rice and Job's Tear mixture, and cooked grain diet (CGD)(autoclaved RGD at 121$^{\circ}C$ for 3 hours). Feeding UCD for 5 weeks significantly lowered the food efficiency ratio (FER) of rats than the value for control animals, and dietary supplementation of brown rice and Job's Tear mixture to UCD significantly restored the FER. Serum total cholesterol concentration was significantly lowered in rats fed RGD (24% decrease) or CGD (16% decrease) compared to the value for rats fed UCD. Feeding RGD for 5 weeks significaly lowered the serum LDL+VLDL-cholesterol concentration (26% decrease), as well as the hepatic cholesterol level (16% decrease) than the values for UCD rats. Animals fed CGD (38% decrease) or RGU (59% decrease) showed significantly lower level of hepatic thiobarbituric acid reactive substances (TBARS) compared to the value for rats fed UCD (p<0.05), although hepatic activities of antioxidative enzymes were not influenced by dietary supplementation. Feeding RGD for 5 weeks significantly increased CD4$^{＋}$ T-cell population along with CD4$^{＋}$/CD8$^{＋}$ ratio of mesenteric lymph nodes compared to those for UCD rats (p<0.05). In conclusion, dietary supplementation of brown rice and Job's Tear mixture as raw grains exhibited superior activity lowering blood and hepatic levels of cholesterol, and improving mesenteric lymph nodes immune function of rats to the cooked grain mixture of identical ingredients.