• Journal of Life Science
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• v.19 no.9
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• pp.1226-1231
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• 2009

Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

• Journal of Magnetics
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• v.20 no.4
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• pp.381-386
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• 2015

The purpose of this study is to improve diagnostic efficiency of clinical study by setting up guidelines for more precise examination with a comparative analysis of signal intensity and image distortion depending on the location of X axial of object when performing magnetic resonance diffusion weighted imaging (MR DWI) examination. We arranged the self-produced phantom with a 45 mm of interval from the core of 44 regent bottles that have a 16 mm of external diameter and 55 mm of height, and were placed in 4 rows and 11 columns in an acrylic box. We also filled up water and margarine to portrait the fat. We used 3T Skyra and 18 Channel Body array coil. We also obtained the coronal image with the direction of RL (right to left) by using scan slice thinkness 3 mm, slice gap: 0mm, field of view (FOV): $450{\times}450mm^2$, repetition time (TR): 5000 ms, echo time (TE): 73/118 ms, Matrix: $126{\times}126$, slice number: 15, scan time: 9 min 45sec, number of excitations (NEX): 3, phase encoding as a diffusion-weighted imaging parameter. In order to scan, we set b-value to $0s/mm^2$, $400s/mm^2$, and $1,400s/mm^2$, and obtained T2 fat saturation image. Then we did a comparative analysis on the differences between image distortion and signal intensity depending on the location of X axial based on iso-center of patient's table. We used "Image J" as a comparative analysis programme, and used SPSS v18.0 as a statistic programme. There was not much difference between image distortion and signal intensity on fat and water from T2 fat saturation image. But, the average value depends on the location of X axial was statistically significant (p < 0.05). From DWI image, when b-value was 0 and 400, there was no significant difference up to $2^{nd}$ columns right to left from the core of patient's table, however, there was a decline in signal intensity and image distortion from the $3^{rd}$ columns and they started to decrease rapidly at the $4^{th}$ columns. When b-value was 1,400, there was not much difference between the $1^{st}$ row right to left from the core of patient's table, however, image distortion started to appear from the $2^{nd}$ columns with no change in signal intensity, the signal was getting decreased from the $3^{rd}$ columns, and both signal intensity and image distortion started to get decreased rapidly. At this moment, the reagent bottles from outside out of 11 reagent bottles were not verified from the image, and only 9 reagent bottles were verified. However, it was not possible to verify anything from the $5^{th}$ columns. But, the average value depends on the location of X axial was statistically significant. On T2 FS image, there was a significant decline in image distortion and signal intensity over 180mm from the core of patient's table. On diffusion-weighted image, there was a significant decline in image distortion and signal intensity over 90 mm, and they became unverifiable over 180 mm. Therefore, we should make an image that has a diagnostic value from examinations that are hard to locate patient's position.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.447-463
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• 2007

Periodontal ligament (PDL) cells have been known as multipotential cells, and as playing an important rolesin periodontal regeneration. The PDL cells are composed of heterogeneous cell populations which have the capacity to differentiate into either cementoblasts or osteoblasts, depending on needs and conditions. Therefore, PDL cells have the capacity to produce mineralized nodules in vitro in mineralization medium which include ascorbic acid, ${\beta}$-glycerophosphate and dexamethasone. In spite of these well-known osteoblast like properties of PDL cells, very little is known about the molecules involved in the formation of the mineralized nodules in the PDL cells. In the present study, we analysed gene-expression profiles during the mineralization process of cultured PDL cells by means of a cDNA microarray consisting of 3063 genes. Nodules of mineralized matrix were strongly stained with alizarin red S on the PDL cells cultured in the media with mineralization supplements. Among 3,063 genes analyzed, 35 were up-regulated more than two-fold at one or more time points in cells that developed matrix mineralization nodules, and 38 were down-regulated to less than half their normal level of expression. In accord with the morphological change we observed, several genes related to calcium-related or mineral metabolism were induced in PDL cells during osteogenesis, such as IGF-II and IGFBP-2. Proteogycan 1, fibulin-5, keratin 5, ,${\beta}$-actin, ${\alpha}$-smooth muscle actin and capping protein, and cytoskeleton and extracellular matrix proteins were up-regulated during mineralization. Several genes encoding proteins related to apoptosis weredifferentially expressed in PDL cells cultured in the medium containing mineralization supplements. Dkk-I and Nip3, which are apoptosis-inducing agents, were up-regulated, and Btf and TAXlBP1, which have an anti-apoptosis activity, were down-regulated during mineralization. Also periostin and S100 calciumbinding protein A4 were down-regulated during mineralization.

• Journal of Life Science
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• v.19 no.1
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• pp.101-110
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• 2009

The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel $mas2^+$ (mitosis associated protein) gene, a homolog of human SMARCAD1 was isolated and characterized from a fission yeast Schizosaccharomyces pombe (S. pombe) using gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 922 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that an SNF2 domain is located, which is involved in the chromosome remodeling. The quantitative analysis of the $mas2^+$ transcript against $adh1^+$ showed that the expression level of $mas2^+$ is high before septum formation in S. pombe. When $mas2^+$ null mutant cells were grown at 27 and $35^{\circ}C$, the cytokinesis of $mas2^+$ null mutant was greatly delayed and a large number of multi-septate and mis-segregated cells were produced. In addition, the number of multi-septate cells significantly increased. When cells were cultured in YES rich medium to increase proliferation, the abnormal phenotypes $mas2^+$ null mutant dramatically increased. These phenotypes could be rescued by an over-expression of the mast gene. The Mas2 protein localized in the nuclei of S. pombe, as evidenced by Mas2-EGFP signals. These results suggest that the $mas2^+$ is homologous to human SMARCAD1 gene and involved in septum formation and chromosome remodeling control.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.277-285
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• 2006

The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.286-290
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• 2009

The transcription of mRNA of avian influenza virus is regulated temporally during infection. Therefore, the measurement of transcript level in host cells should be performed before viral release from host cells because errors can occur in the analysis of the transcript levels if the viruses released from the infected cells re-infect cells. In this study, the timing of viral release was determined by measuring the level of viral RNA from viruses released from H9N2-infected chicken fibroblast cell line UMNSAH/DF-1 by semi-quantitative RT-PCR. The viral genomic RNA was isolated together with mouse total RNA which was added to the collected medium as carrier to monitor the viral RNA recovery and to use its GAPDH as an internal control for normalizing reverse transcription reaction as well as PCR reaction. It was found that viral release of H9N2 in the chicken fibroblast cell line UMNSAH/DF-1 took between 16 and 20 h after infection. We measured all 8 viral mRNA levels. Of the 8 transcripts, 7 species of viral mRNAs (each encoding HA, NA, PB1, PB2, NP, M, NS, respectively) except PA mRNA showed robust amplification, indicating these mRNA can be used as targets for amplification to measure transcript levels. These results altogether suggest that the method in this study can be used for screening antiviral materials against viral RNA polymerase as a therapeutic target.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Journal of Life Science
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• v.27 no.11
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• pp.1276-1289
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• 2017

This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.

• Tuberculosis and Respiratory Diseases
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• v.54 no.4
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• pp.439-448
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• 2003

Background : Surfactant protein B(SP-B) and surfactant protein C(SP-C) are important in accelerating surface spreading of surfactant phospholipid. The glucocorticoids accelerate the morphologic differentiation of epithelial cells into type II cells and increase the rate of phosphatidylcholine synthesis. The hydrophobic surfactant protein has been shown to be upregulated by glucocorticoids in vitro, however, its regulation in vivo is not well established. Methods : The authors investigated the effects of glucocorticoid on the accumulation of mRNA encoding SP-B and SP-C protein content of the lung. Adult rats were given different doses of subcutaneous dexamethasone and sacrificed at 24 hours and 1 week. SP-B and SP-C mRNA were measured by a filter hybridization method. Results : 1) The accumulation of SP-B mRNA at 24 hours after 0.2 mg/kg dexamethasone treatment was increased by 23.7%. 2) The accumulation of SP-B mRNA at 1 week after 2 mg/kg dexamethasone treatment was significantly increased by 96.6%(P<0.001). 3) The accumulation of SP-C mRNA at 24 hours after 0.2 mg/kg dexamethasone treatment was significantly increased by 42.7%(P<0.01). 4) The accumulation of SP-C mRNA at 1 week after 2 mg/kg dexamethasone treatment was significantly increased by 60.0% (P<0.01). Conclusion : The authors concluded that dexamethasone treatment in vivo resulted in increased levels of SP-B mRNA and SP-C mRNA. These results suggested that dexamethasone stimulates the synthesis of hydrophobic proteins associated with surfactant.