• Journal of Life Science
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• v.11 no.2
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• pp.111-115
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• 2001

This study was designed to clone the SNF2/SW12 helicase-related genes from the fission yeast Schizosaccha-romyces pombe and thereafter to elucidate the common functions of the proteins in this family. The $hrp^{2+}$gene was cloned by polymerase chain reaction amplification using degenerative primers from conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. Like other SNF2/SW12 family proteins, the deduced amino acid sequence of Hrp2 contains DNA-dependent ATPase/7 helicase domains as well as the chromodomain and the DNA binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-dinding protein 1), suggesting that Hrp2 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to control the gene expression. To characterize the function of Hrp2, 4 Uracil-Hrp2 fusion protein, it was purified near homogeneity by affinity chromatography on $Ni^{2+}$-NTA agarose, DEAE-Sepharose ion exchange arid Sephacryl S-200 gel filtration chromatographies. The purified fusion protein exhibited DNA-dependent ATPase activity, which was stimulated by both double-stranded and single-stranded DNA. To determine the steady-state level of $hrp^{2+}$ transcripts during growth, cells were cultured in medium and collected at every 2hr to prepare total RNAs. The northern blot analysis showed that the level of $hrp^{2+}$ transcripts reached its maximum before the cells entered the exponential growth phase and then decreased gradually, This result implies that Hrp2 may be required at early stages of cell growth.h.

• KSBB Journal
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• v.13 no.3
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• pp.231-237
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• 1998

An attenuated Japanese encephalitis virus (JEV) clone SA-14-14-2(Vero) was obtained through successive adaptation of a primary cell adapted strain, SA-14-14-2(PDK) to Vero cell, a continuous line of monkey kidney cells. The virus titer reached above the 107 plaque forming unit (pfu) per mL of culture supernatant with 3 passages in Vero cells and was maintained close to this level in the further passages. The optimum temperature for the virus growth was $35^{\circ}C$. Growth pattern of the virus indicated that optimum time for the virus harvest is 4 days post infection and the virus accomplished rapid initial propagation even in medium containing no serum supplement. The roller bottle (RB) system and the spinner flask (SF) system using micro-carrier (Cytodex-1) for the JEV cultivation were explored. When RB, SF, and T-flask culture system were compared, there was no significant difference in virus yield. Furthermore, the results indicated that virus could be harvested multiple times from 3 days to 9 days post infection; neither severe cytopathic effect (CPE) in the infected cells nor the decrease in the titer was observed on duration of 9 days.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.829-835
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• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.

• International Journal of Stem Cells
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• v.17 no.3
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• pp.309-318
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• 2024

Nucleotide-binding oligomerization domain 1 (NOD1), a cytosolic pattern recognition receptor protein, plays a crucial role in innate immune responses. However, the functional expression of NOD1 in mesenchymal stem cells (MSCs) derived from endometriosis remains unclear. The aim of this study was to explore the functions of NOD1 in ectopic endometrial lesions. Tissues and MSCs were isolated from both normal endometrium and endometriosis. Immunohistochemistry and real time quantitative polymerase chain reaction (RT-qPCR) were used to determine the expression of NOD1 in the tissues/MSCs. Quantification of various cytokines was performed using RT-qPCR and enzyme-linked immunosorbent assay. To confirm the proliferation, invasion/migration, and apoptotic viabilities of the samples, Cell Counting Kit-8, clonogenic formation, transwell assays, and apoptotic experiments were conducted. Higher levels of NOD1 expression were detected in the ectopic-MSCs obtained from endometriosis compared to those from the endometrium. The expression of interleukin-8 was higher in the ectopic-MSCs than in the eutopic-MSCs. Pretreatment with NOD1 agonist significantly enhanced the proliferation and invasion/migration of eutopic-MSCs. Additionally, the NOD1 inhibitor ML-130 significantly reduced the proliferation, clone formation, invasion, and migration abilities of the ectopic-MSCs, having no effect on their apoptosis capacity. Our findings suggest that the expression of NOD1 in ectopic-MSCs may contribute to the progression of ectopic endometrial lesions.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.4
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• pp.253-263
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• 2006

Objective: Identification of the regulatory mechanism for arrest and initiation of primordial follicular growth is crucial for female fertility. Previously, we found 15 expressed sequence tags (ESTs) that were specifically abundant in the day-S-subtracted cDNA library and that the B357 clone was novel. The present study was conducted to obtain the whole sequence of the novel gene including B357 and to characterize its mRNA and protein expression in mouse ovary and testis. Methods: The extended sequence of the 2,965-bp cDNA fragment for the clone B357 was named ${\underline{5}}-{\underline{d}}ay-{\underline{o}}vary-{\underline{s}}pecific\;gene-{\underline{1}}$ (5DOS1) and submitted to GenBank (accession number ${\underline{AY751521}}$). Expression of 5DOS1 was characterized in both female and male gonads at various developmental stages by Northern blotting, real-time RT-PCR, in situ hybridization, Western blotting, and immunohistochemistry. Results: The 5DOS1 transcript was highly expressed in the adult testis, brain, and muscle as compared to the other tissues. In the ovary, the 5DOS1 transcript was detected in all oocytes from primordial to antral follicles, and highly expressed at day 5 after birth and decreased thereafter. In contrast, expression of 5DOS1 showed a gradual increase during testicular development and its expression was limited to various stages of male germ cells except spermatogonia. Conclusions: This is the first report on the expression and characterization of the 5DOS1 gene in the mouse gonads. Further functional analysis of the 5DOS1 protein will be required to predict its role in gametogenesis.

• Journal of Veterinary Clinics
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• v.30 no.4
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• pp.292-295
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• 2013

A 9-year-old, male, Doberman pinscher dog with 5-month history of intermittent hematuria, vomiting and glucosuria was referred to local animal hospital. Abdominal ultrasonography showed an irregular and hyperechoic mass in the renal medulla of the enlarged left kidney. Grossly atrophied renal cortex and medulla and marked hydronephrosis were observed on the cut surface of kidney. A single, numerous papillary projected, pedunculated mass 4~5.5 cm in diameter was occupied in renal pelvis, and extended from pelvis to the inlet of ureter. Histopathologically, the mass had numerous papillary structures with arboriform pattern. These papillae were consisted of fibro-vascular stalks that were lined by multiple layers of neoplastic urothelium (transitional epithelium) with marked cellular atypia. Immnohistochemical (IHC) staining demonstrated that the neoplastic cells showed strong positive reactions for cytokeratin (CK) 7, CK 19, CK clone MNF116 and CK high molecular weight, but negative signals for CK 8 low molecular weight. Based on the gross findings, histopathology and CKs profile using IHC staining, this mass was diagnosed as renal pelvis transitional cell carcinoma in a dog.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.201-209
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• 2007

The CFDA (6-carboxyfluorescein diacetate) direct viable count method and plate count (PC) method using conventional nutrient broth (NB) medium and $10^{-2}$ diluted NB (DNB) medium were applied to samples collected from Mt. Yongdoo In Andong, in an effect to determine the number of living bacteria pine mushroom forest soil. The number of living bacteria determined via plate count in NB medium comprised $5{\sim}8%$ of the CFDA direct viable count, and the bacteria in the DNB medium comprised $40{\sim}47%$. This result indicated that viable but nonculturable (VBNC) bacteria existed in the pine mushroom forest soil at a high percentage. The phylogenetic characteristics of the VBNC bacterial populations in the samples of pine mushroom (Tricholoma matsutake) forest soil were analyzed via the direct extraction of DNA and 16S rDNA-ARDRA. The 115 clones from pine mushroom forest soil were clustered into 31 different RFLP phylotypes by ARDRA. Based on the 16S rDNA sequences, the 31 ARDRA clusters were classified into 6 phylogenetic groups: ${\alpha}-,\;{\beta}-,\;{\gamma}-Proteobacteria$, Acidobacteria, Actinobacteria and Firmicutes. Among these bacterial populations, approximately 85% were classified as members of phylum Acidobacteria. The Acidobacteria phylum was shown to exist abundantly in the pine mushroom forest soil.

• Journal of Life Science
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• v.16 no.6
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• pp.904-910
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• 2006

Dendritic cells (DCs) are the only antigen presenting cells (APCs) capable of initiating immune responses, which is crucial for priming the specific cytotoxic T lymphocyte (CTL) response and tumor immunity. Upon activation by DCs, CD4+ helper T cells can cross-prime CD8+ CTLs via IL-12. However, recently activated DCs were described to prime in vitro strong T helper cell type 1 $(Th_1)$ responses, whereas at later time points, they preferentially prime $Th_2$ cells. Therfore, we examined in this study the optimum kinetic state of DCs activation impacted on in vivo priming of tumor-specific CTLs by using ovalbumin (OVA) tumor antigen model. Bone-marrow-derived DCs showed an appropriate expression of surface MHC and costimulatory molecules after 6 or 7-day differentiation. The 6-day differentiated DCs pulsed with OVA antigen for 8 h (8-h DC) and followed by restimulation with LPS for 24 h maintained high interleukin (IL)-12 production potential, accompanying the decreased level in their secretion by delayed re-exposure time to LPS. Furthermore, immunization with 8-h DC induced higher intracellular $interferon(IFN)-{\gamma}+/CD8+T$ cells and elicited more powerful cytotoxicity of splenocytes to EG7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with 24-h DC. In the animal study for the evaluation of therapeutic or protective antitumor immunity, immunization with 8-h DC induced an effective antitumor immunity against tumor of EG7 cells and completely protected mice from tumor formation and prolonged survival, respectively. The most commonly used and clinically applied DC-based vaccine is based on in vitro antigen loading for 24 h. However, our data indicated that antigen stimulation over 8 h decreased antitumor immunity with functional exhaustion of DCs, and that the 8-h DC would be an optimum activation state impacted on in vivo priming of tumor-specific CTLs and subsequently lead to induction of strong antitumor immunity.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.19-24
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• 2005

The low density lipoprotein receptor-related protein 5 (LRP5) highly expressed in many tissues, including hepatocytes and pancreatic beta cells, can bind to apolipoprotein E. To evaluate in vivo roles of LRP5, we generated LRP5-deficient mice. LRP5 genomic DNA was isolated from TT2 embryonic stem (ES) cells. Targeting vector was constructed to disrupt an exon 18 of the mouse LRP5 gene and transfected into ES cells. Three homologous recombinants at LRP5 locus were identified from 178 G418-resistant clones. Chimeric males generated by morula aggregation technique were mated to C57BL/6 female mice. After achieving germ-line transmission, LRP5+/- females were crossed with LRP5+/- males to obtain LRP5-deficient mice. One line of mice lacking LRP5 gene was confirmed by Southern blotting. Such knock-out mice may serve as an effective animal model to study in vivo function of LRP5 gene.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2001.10a
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• pp.61-86
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• 2001

All cancers are caused by abnormalities in DNA sequence. Throughout life, the DNA in human cells is exposed to mutagens and suffers mistakes in replication, resulting in progressive, subtle changes in the DNA sequence in each cell. Since the development of conventional and molecular cytogenetic methods to the analysis of chromosomal aberrations in cancers, more than 1,800 recurring chromosomal breakpoints have been identified. These breakpoints and regions of nonrandom copy number changes typically point to the location of genes involved in cancer initiation and progression. With the introduction of molecular cytogenetic methodologies based on fluorescence in situ hybridization (FISH), namely, comparative genomic hybridization (CGH) and multicolor FISH (m-FISH) in carcinomas become susceptible to analysis. Conventional CGH has been widely applied for the detection of genomic imbalances in tumor cells, and used normal metaphase chromosomes as targets for the mapping of copy number changes. However, this limits the mapping of such imbalances to the resolution limit of metaphase chromosomes (usually 10 to 20 Mb). Efforts to increase this resolution have led to the "new"concept of genomic DNA chip (1 to 2 Mb), whereby the chromosomal target is replaced with cloned DNA immobilized on such as glass slides. The resulting resolution then depends on the size of the immobilized DNA fragments. We have completed the first draft of its Korean Genome Project. The project proceeded by end sequencing inserts from a library of 96,768 bacterial artificial chromosomes (BACs) containing genomic DNA fragments from Korean ethnicity. The sequenced BAC ends were then compared to the Human Genome Project′s publicly available sequence database and aligned according to known cancer gene sequences. These BAC clones were biotinylated by nick translation, hybridized to cytogenetic preparations of metaphase cells, and detected with fluorescein-conjugated avidin. Only locations of unique or low-copy Portions of the clone are identified, because high-copy interspersed repetitive sequences in the probe were suppressed by the addition of unlabelled Cotl DNA. Banding patterns were produced using DAPI. By this means, every BAC fragment has been matched to its appropriate chromosomal location. We have placed 86 (156 BAC clones) cytogenetically defined landmarks to help with the characterization of known cancer genes. Microarray techniques would be applied in CGH by replacement of metaphase chromosome to arrayed BAC confirming in oncogene and tumor suppressor gene: and an array BAC clones from the collection is used to perform a genome-wide scan for segmental aneuploidy by array-CGH. Therefore, the genomic DNA chip (arrayed BAC) will be undoubtedly provide accurate diagnosis of deletions, duplication, insertions and rearrangements of genomic material related to various human phenotypes, including neoplasias. And our tumor markers based on genetic abnormalities of cancer would be identified and contribute to the screening of the stage of cancers and/or hereditary diseases