• Development and Reproduction
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• v.10 no.2
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• pp.85-92
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• 2006

Progesterone(P4) is an important intermediate in the synthesis of androgens and estrogens. Furthermore, P4 itself plays a crucial role in ovulation, atresia and luteinization, and is essential for the continuation of early pregnancy in all mammalian species. In spite of the hormone's physiological importance, the exact action mechanism(s) of P4 in mammalian ovary has not been fully understood yet. In this context, a decades-long controversy regarding the identity of receptors that mediate non-genomic, transcription-independent cellular responses to P4 is presently attracting huge scientific interests. P4 may exert its action in mammalian ovary by several ways: 1) the well-documented genomic pathway, involving hormone binding to so-called classic cytosolic receptor(PGR) and subsequent modulation of gene expression by the ligand-receptor complex as transcription factor. 2) pathways are operating that do not act on the genome, therefore refered to as non-genomic actions. The prominent characteristics of the non-genomic P4 actions are: (i) rapid, (ii) insensitive to transcription inhibitors, (iii) transduced by membrane associated molecules. In particular, the non-genomic P4 actions could be mediated by: (a) classic genomic P4 receptor(PGR) that localizes at or near the plasma membrane, (b) a family of membrane progestin receptors(MPR $\alpha$, MPR $\beta$ and MPR $\gamma$), (c) progesterone receptor membrane component I(PGRMC1), and (d) a membrane complex composed of serpine I mRNA binding protein(SERBP1). The present review summarized these rapid signaling pathways of P4 in the mammalian ovary.

• Animal cells and systems
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• v.5 no.3
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• pp.225-229
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• 2001

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand cellular processes in which Nek2 participates during mammalian development, we investigated the expression and subcellular localization of Nek2 in vivo. The Nek2 protein was detected in spermatocytes and in a fraction of actively dividing ovarian follicle cells and of embryonic tissues. We also observed that Nek2 was localized in both the nucleus and centrosome in embryonic cells. Such localization pattern supports the proposal that Nek2 is a mitotic regulator that is involved in multiple cell cycle events during mammalian development.

• 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.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.1989-1996
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• 2015

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1℃/min in a -80℃ freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.184-193
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• 2007

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

• Development and Reproduction
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• v.1 no.2
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• pp.189-202
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• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.

• BMB Reports
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• v.39 no.5
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• pp.511-515
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• 2006

Alanine aminotransferase (AAT) is mainly synthesized in the liver, and its level in mammalian serum is elevated after acute phase induction. Here we demonstrated that sheep anti-human AAT antibody cross-reacted with amphioxus humoral fluids as well as human serum; and the concentration of AAT in the humoral fluids in amphioxus increased after the acute challenge with lipopolysaccharide, while the level of total proteins remains unchanged. These suggest the presence of the same acute phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AAT was localized in the hepatic diverticulum, ovary and testis. It appears that the hepatic diverticulum in amphioxus is functionally homologous to the vertebrate liver in respect of AAT synthesis, supporting the hypothesis that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.189-189
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• 2001

For a continuous expression of human cytochrome p450 3A5 (CYP3A5) and NADPH-cytochrome P450 reductase (CYPR) proteins, bicistronic construct (CYP3A5BC-LNCX2) was made using internal ribosome entry site (IRES). As for mammalian cell expression, we used pLNCX2 retroviral vector; and using calcium phosphate, plasmid transfer was achieved in 293GPG cell and transduced in Chinese hamster ovary (CHO) cell.(omitted)

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.810-815
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• 2004

Many biological properties and the clinical potential of human interleukin-2 (hIL-2) draw much attention to its high-level expression in mammalian cells. Recombinant human IL-2 (rhIL-2) was expressed in Chinese hamster ovary (CHO) cells, using the recently developed expression system which confers position-independent expression. Stable CHO cell lines carrying several hundred amplified copies of the rhIL-2 gene were easily obtained and rhIL-2 was expressed at high levels after selection with increasing concentrations of methotrexate. Interestingly, the insertion of the transcription terminator of the human gastrin gene into the downstream region of the gene for rhIL-2 considerably increased rhIL-2 expression. Using the expression system with the transcription terminator, it was possible to get a CHO cell line expressing the rhIL-2 at a very high level, about $11.4\mug/10^6$ cell/day, which is about 6 times higher than that previously reported. The biological activity of the rhIL-2 protein purified from the cell line was also confirmed by the cell proliferation assay.

• Korean journal of applied entomology
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• v.43 no.2
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• pp.155-162
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• 2004

Serotonin receptor binds to serotonin (5-HT) and activates effector proteins such as adenylyl cyclase, phospholipase C, cyclic GMP phosphodiesterase or ion channel through G protein on the cell membrane, resulting in various physiological responses like diuresis, memory and development. To examine the comparative expression of the 5-HT$\_$7/ receptor of Aedes aegypti, the Aedes 5-HT$\_$7/ receptor gene was transfected into Drosophila Schneider2 (S2) cells and mammalian Chinese hamster ovary (CHO)-Kl cells. The expression of the Aedes 5-HT$\_$7/ receptor gene in selected cell lines, Tr-CHO and Tr-S2, was confirmed with reverse transcription-PCR, Western blot and immunocytochemistry. Compared with the induced intracellular cAMP level of Tr-S2 cell line to 5-HT, the induced cAMP in the Tr-CHO cell line was over 9 times higher and was dose-dependent. These results suggest that the functionality of Aedes 5-HT$\_$7/ receptor is much more effective in mammalian CHO-K 1 cells and that the Tr-CHO cell line expressing Aedes 5-HT$\_$7/ receptor can be used for synthetic agonist or antagonist candidate screening.