• Korean Journal of Microbiology
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• v.46 no.1
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• pp.21-26
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• 2010

As the causative agent of Anthrax, Bacillus anthracis causes an acute fatal disease in herbivores such as cattle, sheep, and horses as well as humans. The therapeutics and prevention of anthrax currently available are based on antibiotics and the live attenuated vaccine strains, which may be problematic due to the emergency of antibiotic resistant strains or residual virulence in those vaccine strains. Therefore, it has been required to develop novel therapeutics and vaccines which are safer and applicable to humans. Recently, the development of the multivalent vaccine targeting both spores and vegetative cells of B. anthracis along with anthrax toxin has been reported. In our attempts to screen potential candidates for those multivalent vaccines, the whole genomic expression library of B. anthracis was constructed in this study. To the end, the partial digests of the genomic DNA from B. anthracis (ATCC 14578) with Sau3AI were ligated with the inducible pET30abc expression vectors, resulting in approximately $1{\times}10^5$ clones in E. coli BL21(DE3). The redundancy test by DNA nucleotide sequencing was performed for the randomly selected 111 clones and found 56 (50.5%) B. anthracis genes, 17 (15.3%) vector sequences, and 38 (34.2%) unknown genes with no sequence homology by BLAST. An inducible expression of the recombinant proteins was confirmed by Western blot. Interestingly, some clones could react with the antiserum against B. anthracis. These results imply that the whole genomic library constructed in this study can be applied for analyzing the immunomes of B. anthracis.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Journal of Life Science
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• v.25 no.1
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• pp.53-61
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• 2015

In this study, we tried to separate the photosensitizer that induces apoptosis of leukemia cells (U937) from perilla leaves. Perilla leaves (Perilla frutescens Britt var. japonica Hara) are a popular vegetable in Korea, being rich in vitamins (A and E), GABA, and minerals. Dried perilla leaves were extracted with methanol to separate the photosensitizer by various chromatographic techniques. The structure of the isolated compound (PL9443) was identified by 1D-NMR, 2D-NMR, and FAB-mass spectroscopy. Absorbance of the UV-Vis spectrum was highest at 410 nm and was confirmed by the 330, 410, and 668 nm. PL9443 compound was determined to be pheophorbide, an ethyl ester having a molecular weight of 620. It was identified as a derivative compound of pheophorbide structure when magnesium comes away from a porphyrin ring. Observation of morphological changes in U937 cells following cell death induced by treated PL9443 compound revealed representative phenomena of apoptosis only in light irradiation conditions (apoptotic body, vesicle formation). Results from examining the cytotoxicity of PL9443 substance against U937 cells showed that inhibition rates of the cell growth were 99.9% with the concentration of 0.32 nM PL9443. Also, the caspase-3/7 activity was 99% against U937 cells with the concentration of 0.08 nM of PL9443 substance. The result of the electrophoresis was that a DNA ladder was formed by the PL9443. The PL9443 compound is a promising lead compound as a photosensitizer for photodynamic therapy of cancer.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.198-203
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• 2007

DNA transfection is a powerful tool for studying gene functions. The $Ca^{2+}$-phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of $Ca^{2+}$-phosphate precipitation under microscope. Cerebral glial cells were grown until ${\sim}70-80%$ confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and 319 hippocampal neurons were plated onto the glial layer Formation of fine $DNA/Ca^{2+}$-phosphate precipitates was induced using Clontech $CalPhos^{TM}$ Mammalian Transfection Kit, and the size ($0.5-1\;{\mu}m$ in diameter) and density(about 10 particles/$100\;{\mu}m^2$) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of $pEGFP-CaMKII{\alpha}$, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.

• Journal of the Korea Organic Resources Recycling Association
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• v.16 no.4
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• pp.64-73
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• 2008

The cDNA of CCF (coelomic cytolytic factor)-like gene (EC 3.2.1.16), a kind of glycosyl hydorlase, was isolated and cloned from the midgut of the earthworm Eisenia anderi. The size of nucleotide sequence appeared to be 1,152 bp and its predicted coding region was composed of 384 amino acid residues including the initiation methionine. The 17 residues at N-terminal end in the deduced amino acid sequence were regarded to be a signal peptide. Based on the amino acid sequence analysis, it appeared that this CCF-like protein could belong to glycosyl hydrolase family 16 (GHF16) and showed a high sequence homology of about 79~99% with CCF and CCF-like proteins from other earthworm species. The CCFs and CCF-like proteins from various earthworm species exhibited a 100% homology in the polysacchride-binding motif and glucanase motif. It has been reported that the CCFs isolated from E. fedita appeared to show a broader pattern recognition specificity than those from other earthworm species because this species resides in decaying organic matter showing very high microbial activity, implying that CCF-like protein isolated in this study from E. andrei might exhibit a broad substrate specificity that is a useful characteristic for industrial application. A phylogenetic analysis using the deduced amino acid sequences of CCF-related proteins through the BLASTX revealed that GHF16 families could be divided into three groups of metazoa, viriplantae and eubacteria subfamily. Subsequently the CCF-related proteins of metazoa subfamily could clearly be subgroup into lophotrochozoan and edysozoan type including a deuterostome origin. Further understanding of the biological properties of E. andrei CCF-like protein should be addressed to regulate the ${\beta}$-D-glucan hydrolysis and production for the industrial uses.

• Journal of Chest Surgery
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• v.43 no.6
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• pp.576-587
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• 2010

Background: Effective decellularization and fixation process is critical, in order to use xenogenic valves clinically. In the present study, we decellularized bovine pericardium using sodium dodecyl sulfate (SDS) and N-lauroyl sarcosinate, treated with $\alpha$-galactosidase, and then fixed in various manners, to find out the most effective tissue preservation & fixation procedure. Material and Method: Bovine pericardium was decellularized with SDS and N-lauroyl sarcosinate, and treated with $\alpha$-galactosidase. Both groups were fixed differently, by varying glutaraldehyde (GA) or EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide)/N-hydroxysuccinamide (NHS) treatment conditions. Thereafter, physical examination, tensile strength test, thermal stability test, cytotoxicity test, pronase test, pronase-ninhydrin test, purpald test, permeability test, compliance test, H&E staining, DNA quantification, and $\alpha$-galactose staining were carried out to each groups. Result: GA fixed groups showed better physical properties and thermal stability than EDC/NHS fixed groups, EDC/NHS-GA dual fixed groups showed better physical properties and thermal stability than EDC/NHS fixed groups, and showed better thermal stability than GA fixed groups. In pronase test and pronase-ninhydrin test, GA fixed groups and EDC/NHS-GA dual fixed groups showed stronger crosslinks than EDC/NHS groups. Permeability and compliance tended to increase in EDC/NHS-GA dual fixed groups, compared to GA fixed groups. But, EDC/NHS-GA dual fixed groups had stronger tensile strength and lower cytotoxicity than GA fixed groups. Conclusion: We have verified that EDC/NHS-GA dual fixation can make effective crosslinks and lower the toxicity of GA fixation. Henceforth, we will verify if EDC/NHS-GA dual fixation can lower calcifications & tissue failure in vivo experiment.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.203-211
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• 2001

This study was carried out to determine sex of porcine embryos produced by in vitro fertilization. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cystein (0.1 mg/ml) and hormonal supplement (10 IU eCG and 10 IU hCG per ml) for 20~22 hrs. They were then cultured in the same medium but without hormonal supplement for additional 20~22 hrs. After culture, cumulus cells were removed and oocytes were co-incubated for 6 hrs with four different concentrations (5$\times$10$^4$, 2.5$\times$ 10$^{5}$ , 5.0$\times$10$^{5}$ and l0$\times$10$^{5}$ ) of porcine sperm. After fertilization, oocytes were transferred into NCSU 23 with 0.4% BSA medium. The cleavage and blastocyst formation rates were evaluated at 48 and 144 hrs, respectively. In this study, the polymerase chain reaction (PCR) was used to determine the sex of porcine embryos in the stage of blastocyst. The PCR was performed using a set of oligonucleotide primers (5‘-TCATGGACCAGGTAGGGAAT-3', 5’-GAAAGACACGTCCTTGGA GA-3') for 491 bp fragment of porcine male-specific DNA sequence. In the flour different sperm concentration (5$\times$10$^4$, 2.5$\times$10$^{5}$ , 5.0$\times$10$^{5}$ and l0$\times$10$^{5}$ ) for fertilization condition, the cleavage rate was 55.95, 67.88, 60.18 and 47.60%, respectivety, and the development rate of blastocysts was 16.03, 20.40, 21.41 and 12.37%, respectively. At 5.0$\times$10$^4$and 2.5$\times$10$^{5}$ of sperm concentrations per ml cleavage rate and development rate of blastocyst were higher than those of 5.0$\times$10$^4$and l0$\times$10$^{5}$ of sperm concentration (P<0.01). The male of porcine embryos was detected at 491 bp by PCR, and 18 of the 31 porcine blastocysts were the male (58.1%) and the rest 13 were the female(41.9%).

• Journal of Plant Biotechnology
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• v.43 no.4
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• pp.450-456
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• 2016

Rhizoctonia leaf blight (large patch) has become a serious problem in Korean lawn grass, which is extremely hard to treat and develops mostly from the roots of lawn grass to wither it away. Rhizoctonia leaf blight (large patch) is caused by Rhizoctonia solani AG2-2 (IV). To develop zoysia japonica with strong disease tolerance against this pathogenic bacterium, ${\beta}-1,3-glucanase$ was cloned from zoysia japonica, which is one of the PR-Proteins known to play a critical role in plant defense reaction. ${\beta}-1,3-glucanase$ is known to be generated within the cells when plant tissues have a hypersensitive reaction due to virus or bacterium infection and secreted outside the cells to play mainly the function of resistance against pathogenic bacteria in the space between the cells. This study utilized the commonly preserved part in the sequence of corn, wheat, barley, and rice which had been researched for their disease tolerance among the ${\beta}-1,3-glucanase$ monocotyledonous plants. Based on the part, degenerate PCR was performed to find out the sequence and full-length cDNA was cloned. E.coli over-expression was conducted in this study to mass purify target protein and implement in vitro activation measurement and antibacterial test. In addition, to interpret the functions of ZjGlu1 gene, each gene-incorporating plant transformation vectors were produced to make lawn grass transformant. Based on ZjGlu1 protein, antibacterial activity test was conducted on 9 strains. As a result, R. cerealis, F. culmorum, R.solani AG-1 (1B), and T. atroviride were found to have antibacterial activity. The gene-specific expression amount in each organ showed no huge difference in the organs based upon the transformant and against 18s gene expression amount.

• Journal of Applied Biological Chemistry
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• v.59 no.1
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• pp.31-36
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• 2016

Chlorine dioxide has been used for a disinfectant by exhibiting antimicrobial activity and is also potent to kill insect pests infesting stored grains. This study aimed to extend the usefulness of chlorine dioxide with respect to anticancer and antiviral activities. Cytotoxicity of chlorine dioxide was assessed against five different human cancer cell lines. Chlorine dioxide exhibited significant cytotoxicity against two breast cancer cell lines (MCF-7, MDA-MB-231) and three colorectal cancer cell lines (LoVo, HCT-116, SW-480). This cytotoxicity appeared to be associated with the capacity of chlorine dioxide to induce the production of reactive oxygen species (ROS). Compared to control insect cell lines, the cancer cell lines possessed much higher levels of ROS. On the other hand, a treatment of an antioxidant, vitamin E, significantly reduced the cytotoxicity, suggesting that the cytotoxicity was induced by high levels of ROS production. Chlorine dioxide exhibited antiviral activity against different viruses. A baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), is a dsDNA insect virus and lost its viral activity to form polyhedral viral particles in response to chlorine dioxide. The antiviral activity against AcNPV was dependent on the incubation time with chlorine dioxide. Tobacco mosaic virus is a ssRNA plant virus and was reduced in its population after exposure to chlorine dioxide along with significant decrease of viral symptoms. These results indicate that chlorine dioxide possesses anticancer and antiviral activities probably due to its inducing activity of ROS production.

• Journal of Preventive Medicine and Public Health
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• v.36 no.2
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• pp.93-100
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• 2003

Objectives : Because shipyard workers are involved with various manufacturing process, they are exposed to many kinds of hazardous materials. Welders especially, are exposed to bisphenol-A (BPA) during the welding and flame cutting of coated steel, This study was conducted to assess the exposure status of the endocrine disrupter based on the job-exposure matrix. The effects of the genetic polymorphism of xenobiotic enzyme metabolisms involved in the metabolism of BPA on the levels of urinary metabolite were investigated. Methods : The study population was recruited from a shipyard company in the f province. A total of 84 shipbuilding workers 47 and 37 in the exposed and control groups, respectively, were recruited for this study. The questionnaire variables included, age, sex, use of personal protective equipment, smoking, drinking and work duration. The urinary metabolite was collected in the afternoon and correction made for the urinary creatinine concentration. The of the CYP1A1, CYP2E1 and UGT1A6 genotypes were investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods with the DNA extracted from venous blood. Results : The urinary BPA level in the welders group was significantly higher than in the control group (p<0.05). The urinary BPA concentration with the wild type UGT1A6 was higher than the other UGT1A6 genotypes, but with no statistical significant. From themultiple regression analysis of the urinary BPA, the regression coefficient for job grade was statistically significant (p<0.05). Conclusions : The grade of exposure to BPA affected the urinary BPA concentration was statistically significant. However, the genetic polymorphisms of xenobiotics enzyme metabolism were not statistically significant. Further investigation of the genetic polymorphisms with a larger sample size is needed.