• Biomolecules & Therapeutics
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• v.6 no.2
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• pp.191-198
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• 1998

Vsrio vulnificus is an estuarine gram-negative human pathogen that affects people with chronic hepatitis, alcoholic cirrhosis, diabetes mellitus or other underlying diseases. V. vulnificus infection is mediated primarily by consumption of raw fish or by exposure of pre-existing wounds to seawater, causing permanent tissue damages or fatal septic shock. We have been developing a vaccine against V. vulnificus composed of whole cell Iysate of a V. vulnificus O-antigen serotype 4 strain. Oral administration of the V. vulnificus;oral vaccine;immunogenicity;protective efficacy vaccine elicited a high serum antibody response in rabbits. The induced antibodies were reactive not only to the homologous strain but also to heterologous O-antigen serotype strains, indicating cross-reactivities among serotypes. Western blot analysis revealed that the antibodies are mainly specific for outer membrane proteins (OMPs) and reacted equally well with OMPs purified from 9 O-antigen serotypes. The rabbit antisera showed opsonophagocytic killing activity against heterologous strains as well as the homologous strain. Passively transferred rabbit antisera into mice were protective against a lethal V. vulnificus infection. These data demonstrate that oral administration of the V. vulnificus vaccine induced a systemic antibody response which had a protective efficacy against V. vulnificus infections, suggesting that this vaccine preparation could be used to develop an oral vaccine against V. vulnificus.

• Molecules and Cells
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• v.40 no.2
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• pp.143-150
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• 2017

Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

• Korean Journal of Agricultural Science
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• v.46 no.3
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• pp.471-488
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• 2019

Random genes were screened in two transforming ways to investigate the new genes of a ladybug using the Harmonia axyridis cDNA library stock cell cloned in the LITMUS 28i vector in a previous study. Phenotypic variation was observed after injection of the synthesized double-stranded RNA through the in vitro transcription process. The cDNA library of H. axyridis was transformed into E. coli $DH5{\alpha}$ and 10B competent cells by heat shock. Analysis of the nucleotide sequences of the 42 clones with the insert DNAs revealed that 21 clones were homologous with the genes of insects, and only one clone had a gene from H. axyridis. Thirteen of the 21 insect genes were homologous with genes from coleopteran insects. Fourteen genes were selected, which were identified by the gene screening results, and were synthesized as double-stranded RNA through in vitro transcription. One microgram of the synthesized double-stranded RNA between segments T1 and T2 were injected using a syringe into each anesthetized fourth larvae which were under 2 days old. As a result, a phenotypic variation appeared in the larva injected with the two genes. While the eggs of H. axyridis injected with distilled water hatched out three days after oviposition, the eggs of H. axyridis injected with dsHma 06 did not hatch but become shrivel a week after oviposition. Most of the H. axyridis injected with dsHma 08 died and were unable to complete the pupation or eclosion during ecdysis.

• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.248-255
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• 2022

Phlebia sp. MG-60 is the salt-tolerant, white-rot fungus which was isolated from a mangrove forest. This fungus expresses three kinds of manganese peroxidase (MGMnP) isozymes, MGMnP1, MGMnP2 and MGMnP3 in low nitrogen medium (LNM) or LNM containing NaCl. To date, there have been no reports on the biochemical salt-tolerance of these MnP isozymes due to the difficulty of purification. In present study, we established forced expression transformants of these three types of MnP isozymes. In addition, the fact that this fungus hardly produces native MnP in a high-nitrogen medium (HNM) was used to perform isozyme-selective expression and simple purification in HNM. The resulting MGMnPs showed high tolerance for NaCl compared with the MnP of Phanerochaete chrysosporium. It was worth noting that high concentration of NaCl (over 200 mM to 1200 mM) can enhance the activity of MGMnP1. Additionally, MGMnP1 showed relatively high thermo tolerance compared with other isozymes. MGMnPs may have evolved to adapt to chloride-rich environments, mangrove forest.

• BMB Reports
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• v.56 no.2
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• pp.102-107
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• 2023

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

• The Korean Journal of Nuclear Medicine
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• v.25 no.1
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• pp.6-16
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• 1991

Regional cerebral blood flow (rCBF) was evaluated in 12 children ranging in age from 2.7 to 10.0 yr using $^{99m}Tc-HMPAO$ SPECT. For quantitative analysis, 13 pairs of homologous regions of interest (ROIs) were created on three attenuation-corrected 18.8 mm thick transverse slices matching the cerebral cortical regions, deep gray matter, cerebellar hemisphere, and vascular territories, and the semiquantitative indices including "right to left ratio" [(mean count/voxel of homologous right ROI) / (mean count/voxel of homologous left ROI)] and "regional index"(RI) [(mean count/voxel of a ROI)/ (mean count/voxel of all ROIs of each hemisphere)] were calculated. Mean values of right to left ratios of homologous regions ranged from 0.984 to 1.028 in children under 5 yr (group 1) and from 0.982 to 1.012 in children between 5 and 10 yr (group 2), and the mean $value{\pm}2S.D.$ for each region did not exceed 11% and 12% in group 1 and group 2, respectively. There were no statistically significant differences between the RIs of the homologous right and left regions. Significant differences of RIs were found both between vascular regions (p<0.0005 for goup 1, and p=0.0001 for goup 2) and between regions of cerebral cortices (p<0.0005 for group 1, and p<0.005 for group 2) with a relatively high value in the occipital cortex and the lower values in the cerebellum and deep gray matter among the regions of cerebral cortices in both groups. There were no significant differences between the RIs of corresponding regions of group 1 and group 2, except a significantly higher value of right deep gray matter in group 2 than in group 1(p=0.0301). The RIs of the superior frontal cortex and deep gray matter showed to be positively correlated with age (superior frontal cortex; right: rs=0.5254, p=0.0814, left : rs=0.5919, p=0.0496/deep gray matter; right: rs=0.8246, p=0.0062, left: rs=0.6266, p=0.0377). The results suggest that the rCBF pattern of children approaches that of adults in an accipito-rostral direction. This time course of rCBF changes is in agreement with behavioral, neurophysiological, and anatomical alterations known to occur in the developing brain.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.453-475
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• 1998

During the 100 years since the initial discovery of meiotic phenomenon many brilliant aspects have been elucidated, but further researches based on light microscopy alone as an experimental tool have been found to have some limits and shortcomings. By the use of electron microscopy and armed with the advanced knowledges on modern genetics and biochemistry it has been possible to applu molecular technology in gaining information on the detailed aspects of meiosis. As synapsis takes place, a three-layered proteinous structure called the synatonemal complex starts to form in the space between the homologous chromosomes. To be more precise, it begins to form along the paired chromosomes early in the prophase I of meiotic division. The mechanism that leads to precise point-by-point pairing between homologous chromocomes division. The mechamism that leads to precise point-by-point pairing between homologous chromosomes remains to be ascertained. Several items of information, however, suggest that chromsome alignment leading to synapsis may be mediated somehow by the nuclear membrane. Pachytene bivalents in eukaryotes are firmly attached to the inner niclear membrane at both termini. This attached begins with unpaired leptotene chromosomes that already have developed a lateral element. Once attached, the loptotene chromosomes begin to synapse. A number of different models have been proposed to account for genetic recombination via exchange between DNA strands following their breakage and subsequent reunion in new arrangement. One of the models accounting for molecular recombination leading to chromatid exchange and chiasma formation was first proposed in 1964 by Holliday, and 30 years later still a modified version of his model is favored. Nicks are made by endomuclease at corresponding sites on one strant of each DNA duplex in nonsister chromatid of a bivalent during prophase 1 of meiosis. The nicked strands loop-out and two strands reassociate into an exchanged arrangement, which is sealed by ligase. The remaining intact strand of each duplex is nicked at a site opposite the cross-over, and the exposed ends are digested by exonuclease action. Considerable progress has been made in recent years in the effort to define the molecular and organization features of the centromere region in the yeast chromosome. Centromere core region of the DNA duplex is flanked by 15 densely packed nucleosomes on ons side and by 3 packed nucleosomes on the other side, that is, 2000 bp on one side and 400 400 bp in the other side. All the telomeres of a given species share a common DNA sequence. Two ends of each chromosome are virtually identical. At the end of each chromosome there exist two kinds of DNA sequence" simple telpmeric sequences and telpmere-associated sequencies. Various studies of telomere replication, function, and behabior are now in progress, all greatly aided by molecular methods. During nuclear division in mitosis as well as in meiosis, the nucleili disappear by the time of metaphase and reappear during nuclear reorganizations in telophase. When telophase begins, small nucleoli form at the NOR of each nucleolar-organizing chromosome, enlarge, and fuse to form one or more large nucleoli. Nucleolus is a special structure attached top a specific nucleolar-organizing region located at a specific site of a particular chromosome. The nucleolus is a vertical factory for the synthesis of rRNAs and the assenbly of ribosome subunit precursors.sors.

• Journal of Chest Surgery
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• v.31 no.1
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• pp.28-31
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• 1998

Homologous blood transfusion entails substantial risks, including allergic reactions, transmission diseases such as hepatitis, acquired immunodeficiency syndrome. Autotransfusion system is a common method of reducing the need for homologous blood transfusion during cardiac operation. Between July 1993 and July 1995, a series of 40 patients undergoing open heart surgery was selected to an autotransfusion group(n=20) or a control group(n=20). The cell saver system(AT1000, Electromedics, Englewood, CO, USA) was employed for autotransfusion. With this system, shed blood in operative field during cardiopulmonary bypass(CPB) and remained blood in cardiotomy reservior after CPB was aspirated by means of a locally heparinized collecting system. After centrifuge salvaged blood, the resulting red cell concentrate reinfused subsequently. The amounts of blood loss were 766.5$\pm$121.3 ml in cell saver group, 770.1$\pm$113.6 ml in control group, and there were no significant differences between two groups(P=NS). The amounts of blood transfused were 2.91$\pm$1.72 units in cell saver group, 4.82$\pm$1.72 units in control group. Composition of processed blood by cell saver was hemoglobin 17.4 gm%, hematocrit 56.4%, RBC 5,780,000/ul, WBC 9,900/ul, and platelet 33,000/ul. There was no complication related to cell saver. Conclusively, cell saver autotransfusion system is safe, effective method for reducing the homologous blood trasfusion in cardiac surgery.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.05a
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• pp.23-36
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• 2000

Japanese encephalitis virus (JEV) is the causative agent of a mosquito-borne encephalitis and is transmitted to human via persistently infected mosquito vectors. Although the virus is known to cause only acute infection, there were reports that showed neurological sequelae, latent infection in peripheral mononuclear cells, and recurrence of the disease after acute encephalitis. Innate resistance of certain cell lines, abnormal SN1 expression of the virus, and anti-apoptotic effect of cullular bcl-2 have been suggested as probable causes of JEV persistence even in the absence of defective interfering (DI) particles. Although possible involvement of DI particles in JEV persistence was suggested, neither has a direct evidence for DI presence nor its molecular characterization been made. Two questions asked in this study are whether the DI virus plays any role in JEV persistent infection if it is associated with and what type of change(s) can be made in persistently infected cells to avoid apoptosis even with the continuous virus replication, DI-free standard stock of JEV was infected in BHK-21, Vero, and SW13 cells and serial high multiplicity passages were performed in order to generate DI particles. There different-sized DI RNA species which were defective in both structural and nonstructural protein coding genes. Rescued ORFs of the DI genome maintained in-frame and the presence of replicative intermediate or replicative form RNA of the DI particles confirmed their replication competence. On the other hand, several clones with JEV persistent infection were established from the cells survived acute infections during the passages. Timing of the DI virus generation during the passages seemed coincide to the appearance of persistently infected cells. The DI RNAs were identified in most of persistently infected cells and were observed throughout the cell maintenance. One of the cloned cell line maintained the viral persistence without DI RNA coreplication. The cells with viral persistence released the reduced but continuous infectious JEV particle for up to 9 months and were refractory to homologous virus superinfection but not to heterologous challenges. Unlike the cells with acute infection these cells were devoid of characteristic DNA fragmentation and JEV-induced apoptosis with or without homologous superinfection. Therefore, the DI RNA generated during JEV undiluted serial passage on mammalian cells was shown to be biologically active and it seemed to be responsible, at least in part, for the establishment and maintenance of the JEV persistence in mammalian cells. Viral persistence without DI RNA coreplication, as in one of the cell clones, supports that JEV persistent infection could be maintained with or without the presence of DI particles. In addition, the fact that the cells with JEV persistence were resistant against homologous virus superinfection, but not against heterologous one, suggests that different viruses have their own and independent pathway for cytopathogenesis even if viral cytopathic effect could be converged to an apoptosis after all.