• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.195-195
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• 2004

To discover germ cell-specific transcripts, we prepared a cDNA library from adult testes of 35-day old mice and subtracted it with mRNA from the testes of juvenile mice. Real-time RT-PCR analysis indicated that 42 cDNA clones in the subtracted library were expressed more intensely in the adult testes than in the juvenile testes. One clone identified by subtraction is expressed preferentially in the late spermatid and is located on chromosome 17E3 in mouse and 2p22 in human. (omitted)

• Development and Reproduction
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• v.7 no.2
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• pp.119-126
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• 2003

In the previous study, we obtained list of differentially expressed genes between postnatal day 1 and day 5 mouse ovaries using suppression subtractive hybridization(SSH) and found that MT Oansposon-like element, clone MTi7(MTi7) was one of the highly expressed genes in the day 5 mouse ovary(Park et at., 2002). Results of in situ hybridization and RNA interference revealed that the expression of MTi7 is oocyte-specific in the ovary and may be involved in the regulation of oocyte maturation(Park et at., 2003). At present, MTi7 sequence has been known only in the mouse. Therefore, the present study was accomplished 1) to identify MTi7 sequence in the other mammalian species, such as bovine, porcine, rat, and human, and 2) to evaluate the flanking sequence of the mouse MTi7 since it has transposon characteristics. Using ovarian cDNAs derived from low different species, we cloned and identified new MTi7 sequence showing a high degree of sequence homology with the mouse MTi7(87∼98%). By using inverse PCR, we found that the mouse MTi7 may intercalated the beta-carotene 15, 15'-monooxygenase(Bcdo) gene and/or serine protease inhibitor, Kunitz Dpe I(Spint 1) gene. By finding the MTi7 sequences in the other mammalian species and the flanking gene of the MTi7 in mouse, it is expected to reveal the role(s) of MTi7 in the oogenesis as well as folliculogenesis in the near future.

• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.283-287
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• 2001

Differential display based on PCR was employed to identify genes expressed during tuber-developing stage of potato (Solanum tuberosum L. cv. Irish Cobbler). An eukaryotic initiation factor 5A (eIF-5A) clone isolated from a cDNA library constructed with developing micro-tuber using a probe of PCR fragment. We isolated three positive clones and ore of them contained open reading frame. This clone revealed high sequence similarity to tomato eIF 5A cDNA. At the DNA level, there is 94.8% identity with the tomato eIF-5A4, whereas at the protein level there is a high identity with 97.5%. The potato eIF 5A clone is 716 bp in length and contains a single open reading frame from 57 to 539 bp, a 56 bp 5'-untranslated region and a 177 bp 3'-untranslated region. The deduced protein composed of 160 amino acid residues, with a predicted molecular mass of 17.4 kD and an estimated pl of 5.5. The sequence of 12 (STSKTGKHGHAK) amino acids among eIF-5A proteins is perfectly conserved from yeast to human. That sequence in potato eIF-5A protein is also conserved at position 46 to 57 amino acid. This region embeds the post-translational modification site of the lysine residue (at the seventh K) to hypusine that is crucial to eIF-5A activity. The northern blot analysis of eIF5A has shown abundant expression, mainly in flower organs (stamen, ovary, petal, sepal), fruit and stolen.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.245-253
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• 2010

The base sequences representing human- and cow-specific 168 rRNA gene markers identified in a T-RFLP analysis were recovered from clone libraries. The human- and cow-specific primers were designed from these sequences and their specificities were analyzed with fecal DNAs from human, cow, and pig. The AllBac primer set showed positive results for all human, cow, and pig samples, whereas the human-specific primer set showed positive result only for the human sample but not for the cow or pig samples. Likewise, the cow-specific primer set showed positive results only for the cow sample but not for the human or pig samples. Real-time PCR assay with these primers was developed for the identification and quantification of fecal pollution in the river water. The human- and cow-specific markers were detected in the order of 9 $\log_{10}$ copies per gram wet feces, which were two orders of magnitude lower than those of total Bacteroidales. For the river water samples, the human-specific marker was detected in $1.7-6.2\;\log_{10}$ copies/100 ml water, which was 2.4-4.9 orders of magnitude lower than those of total Bacteroidales. There was no significant correlation between total Bacteroidales and conventional fecal indicators, but there was a high correlation between Bacteroidales and the human-specific marker. This assay could reliably identify and quantify the fecal pollution sources, enabling effective measures in the watersheds and facilitating water quality management.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.437-442
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• 2015

Aim: To establish a pancreatic cancer stem cell model using human pancreatic cancer cells in nude mice to provide a platform for pancreatic cancer stem cell research. Materials and Methods: To establish pancreatic cancer xenografts using human pancreatic cancer cell line SW1990, nude mice were randomly divided into control and gemcitabine groups. When the tumor grew to a volume of $125mm^3$, they treated with gemcitabine at a dose of 50mg/kg by intraperitoneal injection of 0.2ml in the gemcitabine group, while the mice in control group were treated with the same volume of normal saline. Gemcitabine was given 2 times a week for 3 times. When the model was established, the proliferation of pancreatic cancer stem cells was observed by clone formation assay, and the protein and/or mRNA expression of pancreatic stem cell surface markers including CD24, CD44, CD133, ALDH, transcription factors containing Oct-4, Sox-2, Nanog and Gli, the key nuclear transcription factor in Sonic Hedgehog signaling pathway was detected by Western blot and/or RT-PCR to verify the reliability of this model. Results: This model is feasible and safe. During the establishment, no mice died and the weight of nude mice maintained above 16.5g. The clone forming ability in gemcitabine group was stronger than that of the control group (p<0.01). In gemcitabine group, the protein expression of pancreatic cancer stem cell surface markers including CD44, and ALDH was up-regulated, the protein and mRNA expression of nuclear transcription factor including Oct-4, Sox-2 and Nanog was also significantly increased (P<0.01). In addition, the protein expression of key nuclear transcription factor in Sonic Hedgehog signaling pathway, Gli-1, was significantly enhanced (p<0.01). Conclusions: The pancreatic cancer stem cell model was successfully established using human pancreatic cancer cell line SW1990 in nude mice. Gemcitabine could enrich pancreatic cancer stem cells, simultaneously accompanied by the activation of Sonic Hedgehog signaling pathway.

• Biomedical Science Letters
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• v.7 no.4
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• pp.161-166
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• 2001

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Molecules and Cells
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• v.24 no.3
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• pp.358-363
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• 2007

Swine endothelial cells are commonly used as an in vitro model for studying features of the blood-brain barrier and some hemorrhagic diseases. However, primary cultures of swine cells have finite lifespans. To establish immortalized swine umbilical vein endothelial cells (SUVECs) using human telomerase reverse transcriptase (hTERT), the plasmid pCI-neo-hTERT was transfected into SUVECs by lipofection. Clones were selected for G418 resistance, and positive clones were amplified. One of the clones was cultured for up to 50 passages. Factor VIII-related antigen and CD34 were detected. The immortalized cells shared the properties of normal cells, such as contact inhibition, serum requirement and anchorage dependence. Karyotype analysis revealed that the immortalized cells were in the diploid range. In addition, both in vivo and in vitro assays of tumorigenicity showed no neoplastic transformation. Furthermore, NO, $PGI_2$, and ET-1 concentrations in the transfected cells were normal. These results suggest that the SUVECs immortalized by hTERT retain their original characteristics.

• Biomedical Science Letters
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• v.11 no.1
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• pp.57-61
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• 2005

The number of sites at which a protein can be readily cleaved by a proteolytic enzyme is greatly influenced by its three-dimensional structure. For native, properly-folded proteins both the rate of cleavage and number of sites at which cleavage takes place are usually much less than for the denatured protein. In order to compare the tertiary structure of recombinant HepG2 type glucose transporter with that of its native counterpart in the erythrocyte, the pattern of tryptic cleavage of the protein expressed in insect cell membranes was therefore examined. After 30 minutes digestion, a fragment of approximate Mr 19,000-21,000 was generated. In addition to this, there were two less intensely stained fragments of apparent Mr 28,000 and 17,000. The pattern of labelling was similar up to 2 hours of digestion. However, the fragments of Mr 19,000-21,000 and Mr 17,000 were no longer detectable after 4 hours digestion. The observation of a very similar pattern of fragments yielded by tryptic digestion of the HepG2 type transporter expressed in insect cells suggests that the recombinant protein exhibits a tertiary structure similar if not identical to that of its human counterpart. Also, the endogenous sugar transporter(s) present in Sf21 cells did not bind cytochalasin B, the potent transporter inhibitor. Therefore, the baculovirus/Spodoptera frugiperda (Sf) cell expression system could be very useful for production of large amounts of human glucose transporters, heterologously.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.39-39
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• 2003

Methylation of DNA 5-cytosine in mammalian early embryo affects great deal in nuclear reprogramming and chromatin remodeling of developing embryo. Current efforts to clone and produce cloned animals including transgenic animals face various problems including low birth rate, irregular development, and so on. In this report, cDNA for the one of house keeping methyltransfcrase, Dnmt1 was cloned from bovine somatic tissues and was analyzed for its nucleotide sequences to investigate the structure and function of the gene in bovine early development. Nucleotide sequence of bovine Dnmt1 homologue showed 76.8% identity with that of human Dnmtl and 66.4% with mouse Dnmt1. Translated amino acid sequence showed 88.4% homology with human homologue and 75.8% homology with mouse counterpart. Three types of Dnmt1 are reported in mouse and human, and are likely present in bovine tissues. Understanding of role of Dnmt1 in bovine development may shed a light in the field of animal, especially bovine cloning.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.248-256
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• 1996

The present study was performed to clone and express human poly (ADP-ribose) synthetase (PARS) cDNA in E coli. For these purposes, the CDNA for human poly (ADP-ribose) synthetase, encoding the entire protein, was cloned into pGEM-7Zf(+). The resulting recombinant plasmid pPARS6.1 was restriction enzyme mapped and its identity was confirmed by Southern blot analysis. The pPARS6. 1 contained full-length CDNA of human PARS and the nudeotide sequences were identical with those reported previously. The recombinant protein which migrated as a unique 120 kDa band on 10% SDS-polyacrylamide gels, was identified as PARS by Southwestern blots using nick-translated DNA probes and by activity gels and activity blots using 32 P-NAD as a substrate for poly (ADP-ribose) synthetase (PARS). The signals corresponding to 120 and 98 kDa proteins were obtained following IPTG (0.4 mM) induction of the PARS cDNA cloned into Xba I-digested pGEM-7Zf(+) vector. Nonspecific signals corresponding to 45 and 38 kDa proteins were also shown in both IPTG-induced and noninduced cells. The nonspecific proteins may be products of incomplete translation or proteolytic products of intact PARS.