• BMB Reports
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• v.41 no.10
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• pp.722-727
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• 2008

The present study compared the proteomic characteristics of a low passage number (L-33) and high passage number (H-81) LNCaP cell clone. Marked differences in protein expression were noted in the response of L-33 and H-81 cells to androgens. To investigate if regulation of these proteins was androgen-dependent, expression of the androgen receptor was silenced via small interfering RNA. Consistent with the proteomic data, abrogation of androgen receptor production in H-81 cells resulted in the reversed expression level into L-33 cells compared with non-treated H-81 LNCaP cells. The results clarify the progression into an androgen-independent phenotype.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.06a
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• pp.19-25
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• 2002

Researches on manipulation pluripotent stem cells derived from blastocysts or promordial germ cells (PGCs) have a great advantages for developing innovative technologies in various fields of life science including medicine, pharmaceutics, and biotechnology. Since the first isolation in the mouse embryos, stem cells or stem cell-like colonies have been continuously established in the mouse of different strains, cattle, pig, rabbit, and human. In the animal species, stem cell biology is important for developing transgenic technology including disease model animal and bioreactor production. ES cell can be isolated from the inner cell mass of blastocysts by either mechanical operation or immunosurgery. So, mass production of blastocyst is a prerequisite factor for successful undertaking ES cell manipulation. In the case of animal ES cell research, various protocol of gamete biotechnology can be applied for improving the efficiency of stem cell research. Somatic cell nuclear transfer technique can be applied to researches on animal ES cells, since it is powerful tool for producing clone embryos containing genes of interest. In this presentation, a brief review was made for explaining how somatic cell nuclear transfer technology could contribute to improving stem cell manipulation technology.

• 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.

• The Journal of the Korean Society for Microbiology
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• v.21 no.3
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• pp.399-406
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• 1986

This study was performed to establish human plasmacytoma cell line as the partner cells for producing human hybridoma. Bone marrow cells from a multiple myeloma patient from Seoul National University Hospital, Korea were cultured and established as the cell line, named as HMC-BM4. HMC-BM4 cells were cultivated in RPMI 1640 media containing 8-azaguanine(8-AG; gradually increasing concentration from $1\;{\mu}g/ml$ to $20\;{\mu}g/ml$). 8-AG resistant cells were collected and cloned by limiting dilution. Each clone was divided and tested to die in hypoxanthine, aminopterine and thymidine (HAT) selection media. Finally one clone was selected and named as HMC-AR, which was sensitive to HAT selection media. HMC-AR cells showed typical morphology of plamacytoma in Wright staining. No cell formed the rosette with sheep erythrocytes. Surface membrane $\mu$ heavy chain was detected in 20% of HMC-AR cells and cytoplasmic $\mu$ heavy chain in 90% of them by direct immunofluorescent staining. Ia-like antigen was found in 90% of HMC-AR cells by indirect immunofluorescent staining using anti-Ia-like antigen monoclonal antibody, 1BD9-2. And about $1.0\;{\mu}g/ml$ of human $\mu$ heavy chain was detected in the 3-day culture supernatant of HMC-AR cells. 88% of cells contained 46 chromosomes. Mycoplasma was not detected in HMC-AR cells by Hoechst 33258 staining. This cell line would be used for making hybridomas secreting human monoclonal antibody.

• Journal of Periodontal and Implant Science
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• v.26 no.2
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• pp.448-468
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• 1996

To maintain its functional integrity, bone is continuously remodelled by a process involving resorption by osteoeclasts and formation by osteoblasts, In order to respond to changes in the physical environment or to trauma with the relevant action, this process is strictly regulated by locally synthesized or systemic fators, Prostaglandin $E_2(PGE_2$) is perhaps one of the best studied factors, having been known to affect bone cell function for several decades.$PGE_2$ has both anabolic and catabolic activities. Excess of $PGE_2$ has been implicated in a number of pathological states associated with bone loss in a number of chronic inflammatory conditions such as periodontal disease and rheumatoid arthritis. $PGE_2$ and other arachidonic acid metabolites have been shown to be potent stimulators of osteoclastic bone resorption in organ culture. The anabolic effects of $PGE_2$ were first noticed when an increase in periosteal woven bone formation was seen after the infusion of $PGE_2$ into infants in order to prevent closure of the ductus arteriosus. The cellular basis for the catabolic actions of $PGE_2$ has been well characterized. $PGE_2$increases osteoclast recruitment in bone marrow cell cultures. Also $PGE_2$ has a direct action on osteoclast serving to inhibit activity and can also indirectly activate osteoclast via other cells in the vicinity, presumably osteoblast. The cellular mechanisms for the anabolic actions of $PGE_2$ are not nearly so well understood. The purpose of this paper was to study the effects of $PGE_2$ and dibutyl(DB)cAMP on osteoblastic clone MC3T3El cells and on the generation of osteoclasts from their precursor cells. The effect of $PGE_2$ and DBcAMP on the induction of alkaline phoaphatase(AlP) was investigated in osteoblastic clone MC3T3El cells cultured in medium containing 0.4% fetal bovine serum. $PGE_2$ and DBcAMP stimulated ALP activity and MTT assay in the cells in a dose-dependent manner at concentrations of lO-SOOng/ml. Cycloheximide, protein synthesis inhibitor, inhibited the stimulative effect of $PGE_2$ and DBcAMP on ALP activity in the cells. $PGE_2$also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 500ng/ml. The effect of $PGE_2$ on the generation of osteoclasts was investigated in a coculture system of mouse bone marrow cells with primary osteoblastic cells cultured in media containing 10% fetal bovine serum.After cultures, staining for tartrate-resistant acid phosphatase(TRAP)-marker enzyme of osteoclast was performed. The TRAP(+) multinucleated cells(MNCs), which have 3 or more nuclei, were counted. More TRAP(+) MNCs were formed in coculture system than in control group. $PGE_2(10^{-5}10^{-6}M)$ stimulated the formation of osteoclast cells from mouse bone marrow cells in culture. $PGE_2(10^{-6}M)$ stimulated the formation of osteoclast cells from mouse bone marrow cells in coculture of osteoblastic clone MC3T3E1 cells This results suggest that $PGE_2$ stimulates the differentiation of osteoblasts and generation of osteoclast, and are involved in bone formation, as well as in bone resorption.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.560-567
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• 1998

We have found that Thermoactinomyces vulgaris KFB-Cl00 produces a chitinase. The optimum temperature and pH of the enzyme activity were $55^{\circ}C$ and 6.5. The enzyme was stable after heat treatment at $80^{\circ}C$ for 30 min and stable in acidic and basic conditions (PH 6.0~11.0). The thermostable endo-chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5$\alpha$ as a host strain. The positive clone carrying a recombinant plasmid (PKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was sub cloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.

• Development and Reproduction
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• v.6 no.1
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• pp.7-16
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• 2002

In order to obtain the specific genes of snailfish a subtracted cDNA library was constructed, and analysed by sequencing and GenBank search. Among them C90-171 clone was turned out to be genes showing low homology and nonredundant genes. This novel clone was named Gomsin(C90-171). Gomsin was shown to be intensely expressed in the epithelial cells, some mesenchymal cells, and sheaths of muscle bundles in the result of immunohistochemistry. In the cross reaction assay of Gomsin antibody against various human tissues, the Gomsin was strongly expressed in the ductal and acinar cells of salivary glands, which was similar to the expression patterns of proline-rich proteins(PRPs) of human. The antibody raised against the Gomsin was clearly cross-reacted with human salivary PRPs and also recombinant proteins of human PRPs in the Western blot and immunoprecipitation analysis. Contrast to the salivary PRPs, the Gomsin was not easily degraded in the mixed saliva, but rapidly attacked on the cultured keratocytes in vitro. The simulated protein structure of Gomsin was similar to the whorled pattern of PRPs, even though the amino acid sequence of Gomsin was quite different from those of PRPs. These data suggest that the Gomsin is a characteristic matrix protein in the skin and body of snailfish, which is also utilized for the tissue protection in the similar way to the PRPs of human muco-secretory organs.

• Toxicological Research
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• v.12 no.2
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• pp.271-275
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• 1996

Carcinogenic potential of HPV-16 DNA and NNK in a human keratinocyte cell line was assessed to study effects of viral-chemical interaction. Human cells were transfected with HPV-16 DNA and 6 clonal cell lines were subsequently obtained. Clonal line-3 and 6 at passage 7 showed characteristics of tumor cells such as increases of saturation density, soft-agar colony formation, cell aggregation and foci appearance. Among cells treated with 1$\mu M$, 10$\mu M$, 100$\mu M$ or 1 mM of NNK for 4 weeks, 100$\mu M$ treatment showed most tumorigenic characteristics at passage 7. These results indicate that either HPV-16 or NNK alone is tumorigenic in this in human in vitro model. When cells transfected with HPV-16 were subsequently exposed by 100 uM NNK for 4 weeks, all the clonal cells except clone-1 showed higher levels of tumor cell characteristics than HPV-16 DNA or NNK exposure alone. Clonal line-6, the most tumorigenic cells, showed higher transcriptional level of fibronectin and lower level of TGF-$\beta_1$, as compared to control cells, suggesting that alteration of growth factor or extracellular matrix may play a role in carcinogenesis process induced by HPV-16 and NNK. Taken together, the present study indicates that viral-chemical interactions between HPV-16 DNA and NNK enhance carcinogenic potentials of human cells and implies that smoking among people infected with human papillomavirus may pose an additional risk of causing cancer.

• BMB Reports
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• v.42 no.5
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• pp.310-314
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• 2009

Globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) are the proposed functional receptors for Shiga toxin (Stx). To elucidate the effect of Gb3 content on Stx-induced cytotoxicity in HeLa cells, we cloned HeLa cells and determined the correlation between glycolipids content and Stx-induced cytotoxicity. The 29 HeLa cell clone (HLCC) lines used showed a wide range of sensitivity to Stx, compared to Gb3-rich cells which were more sensitive, showing as little as 20% viability to 100 pg/ml Stx. In contrast, Gb3-deficient cells proved resistant as they were more than 80% viable to 100 ng/ml Stx. Gb3 content in the HLCC lines corresponded with Stxs-induced cytotoxicity as well as Gb3 synthase expression, but no correlation with Gb4 content was noted. These data show that Gb3 content, which is regulated by the expression of Gb3 synthase, determines the sensitivity of HeLa cells toward Stx.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.1036-1040
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• 2007

Sodium butyrate (NaBu) has been used to enhance protein expression levels in mammalian cell culture. To determine the clonal variability of recombinant Chinese hamster ovary (rCHO) cells in response to NaBu addition regarding specific antibody productivity $(q_{Ab})$, three rCHO clones were subjected to different concentrations of NaBu. For all three clones, NaBu addition inhibited cell growth and decreased cell viability in a dose-dependent manner. On the other hand, the enhancing effect of NaBu on $q_{Ab}$ varied significantly among the clones. NaBu addition enhanced the antibody production of only one clone. RT-PCR analysis revealed that the changes in $q_{Ab}$ correlated linearly with those of the mRNA transcription level. Thus, it was concluded that the different enhancing effects of NaBu on protein expression in rCHO cell clones resulted from their different mRNA transcription levels.