• Development and Reproduction
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• v.2 no.1
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• pp.1-8
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• 1998

In mammalian ovary, major portion(>99%) of ovarian follicles undergo atresia. Recent studies have shown that this phenomenon is mediated via GC apoptosis. Ceramide, a product of sphingomyelin hydrolysis, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In the present study, we have examined the effect of ceramide on apoptotic cell death of GC in vitro. GCs were harvested by squeezing the antral follicles from the immature mice (3-4 weeks) and cultured in MEM medium with 10% fetal bovine serum. The cells were treated with various concentrations of ceramide (0 to 50 \mu M)and cultured up to 24 h.Cell death was determined by MTT cell viability assay and apoptosis was examined by acridine orange staining, in situ 3'-end labeling(TUNEL), and flow cytometry. Ceramid treatment induced apoptotic cell death of GC in a time- and a dose-dependent manner. Results of flow cytometric analysis showed that creamide-induced cell death was mostly confined to the $G_{0}$/$G_{1}$ cells. these results provide an evidence for ceramide as a lipid second messenger of apoptosis in mouse GC.

• Toxicological Research
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• v.3 no.1
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• pp.15-26
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• 1987

The mutagenicity of pranoprofen, a new antiinflammatory agent primarily used in Japan, was evaluated by employing several different methods such as the Ames test, micronucleus test, and the sister chromatid exchange test. For the Ames test, various doses of pranoprofen (5 and 1 mg, 100, 10, and 1 ${\mu}$g per plate) were applied, with or without the mammalian liver S-9 fraction, to the S. typhimurium LT2. For the micronucleus test, 24 hours after administering the various doses of pranoprofen (200, 100, and 50 mg/kg) to male mice by aral intubation, the femura of each group were isolated and the bone marrow samples were prepared. The micronucleated red cells and the ratio of the polychromatic versus the normochroomatic cells were counted. For the sister chromatid exchange test, the maximal non-cytotoxic concentrations (10 to 0.1 mM pranoprofen) were applied to the culture media of the Chinese Hamster Ovary (CHO) cells for 24 hrs. The numbers of revertant colonies did not increase with the increasing doses of pranoprofen when teseted with various strains of S. typhimurium. In the micronucleus test employing mice, the pranoprofen was identkfied to be a non-clastogen and a non-spindle poison. In the sister chromatid exchange test employing the cultured CHO cells, the pranoprofen did not increase the incidences of chromosomal abnormality. Based on these results, pranoprofen was found to have no mutagenic activity.

• Parasites, Hosts and Diseases
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• v.55 no.6
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• pp.643-652
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• 2017

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis' gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.217-221
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• 2010

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1~2 mm, 2~6 mm and 6~10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2~6 mm follicle and 5 spots from follicle fluid of 6~10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2~6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6~10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.207-207
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• 1996

Through molecular cloning, five muscarinic receptors have been identified. The muscarinic receptors can be generally grouped according to their coupling to either stimulation of phospholipase C (m1, m3, and m5) or the inhibition of adenylate cyclase (m2 and m4). Each m1, m3, and m5 receptors has the additional potential to couple to the activation of phospholipase A$_2$, C, and D, tyrosine kinase, and the mobilization of Ca$\^$2+/. However, the differences in coupling efficiencies to different second messenger systems between these receptors have not been studied well. Ectopic expression of each of these receptors in mammalian cells has provided the opportunity to evaluate the signal transduction of each in some detail. In this work we compared the coupling efficiencies of the m1, m3 and m5 muscarinic receptors expressed in chinese hamster ovary (CHO) cells to the Ca$\^$2+/ mobilization and the stimulation of neuronal nitric oxide synthase (nNOS). Because G protein/PLC/PI turnover/[(Ca$\^$2+/])i/NOS pathway was supposed as a main pathway for the production of nitric oxide via muscarinic receptors, we studied on ml, m3 and m5 receptors. Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an index of generation nitric oxide (NO) in CHO cells. The agonist carbachol increased the cGMP formation and the intracellular [Ca$\^$2+/] in concentration dependent manner in three types of receptors and the increased cGMP formation was significantly attenuated by scavenger of NO or inhibitor of NOS. m5 receptors was most efficiently coupled to stimulation of nNOS, And, the coupling efficiencies to the stimulation of neuronal nitric oxide synthase in three types of receptors were parallel with them to the Ca$\^$2+/ mobilization.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.177-182
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• 2014

Feline ovulation time after LH surge have not been defined because its LH surge is occurred by several times of coital vaginal induction and cat has relatively longer time between LH surge and ovulation compared with other mammalian species. This study was performed to investigate the feline ovulation time after LH surge that was induced by hCG injection for superovulation with PMSG. For superovulation, all cats were received an initial injection of PMSG (200 IU, i.m.) followed 80 hrs later with an injection of hCG (200 IU, i.m.). And then, sampling of both ovaries was surgically performed at each 6 different times (10, 18, 22, 26, 29, and 32 hrs) after hCG injection. Cumulus-oocyte-complexes (COCs) were collected from 2 sides of oviducts and ovaries were fixed for ovarian histology. Total 38 COCs were collected only at hCG 32 hrs and no COCs were shown at earlier 5 times. However, in the ovarian histology, corpus haemorrhagicum or corpus luteum was not shown in all groups including ovary at hCG 32 hrs that COCs were collected. In conclusion, it was suggested that feline ovulation was occurred at 29~32 hrs after LH surge and taken relatively long time for CL formation after ovulation.

• BMB Reports
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• v.38 no.4
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.255-261
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• 2006

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. Here, we report expression of the DNA methyltransferases (Dnmts) family during mouse early development. Transcription of Dnmt1o occurs in one-cell and morula stage embryos, whereas Dnmtls transcripts were detectable in all cells and tissues examined during the study. Dnmt3a1 transcript was detected in all cells and Dnmt3a2 transcript was particularly detected in the oocyte and 1-cell stages. Low level Dnmt3b1 transcripts were expressed ubiquitously in oocyte, 1-cell, and preimplantation embryos except $2{\sim}4cell$ stages. Dnmt3b3 transcripts were only detected in E7.5 embryo and ovary. Furthermore, Dnmt31 transcripts were detectable in all cells and tissues examined. Unlike Dnmtl, both Dnmt3a and Dnmt3b proteins existed in the nucleus of preimplantation embryos till the morula stage. These Results suggest that differences Dnmts expression level exist and genomic DNA methylation patterns may be determined partly through differential expression of Dnmts during early development.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.5
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• pp.265-272
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• 2010

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and $\geq25mm$). These follicles had granulosa cell layer and theca interna and the hormone $17{\beta}$-estradiol ($E_2$)/ progesterone ($P_4$) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using $GeneFishing^{TM}$ DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.

• Animal cells and systems
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• v.5 no.3
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• pp.253-262
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• 2001

Ligand-receptor clustering event is the most important step in leukocyte adhesion and spreading on endothelial cells. Intercellular adhesion molecule-1 (ICAM-1) has been shown to enhance leukocyte adhesion, but the molecular event during the process of adhesion is unclear. To visualize the dynamics of ICAM-1 movement during adhesion, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 fused to a green fluorescent protein (IC1_GFP/CHO) and examined them under the fluorescence microscopy. The transfection of IC1_GFP alone in these cells was sufficient to support the adhesion of K562 cells that express $\alpha$L$\beta$2 (LFA-1) integrin stimulated by CBR LFA-1/2 mAb. This phenomenon was mediated by ICAM-1-LFA-1 interactions, as an mAb that specifically inhibits ICAM-1-LFA-1 interaction (RRl/l) completely abolished the adhesion of LFA-1＊ cells to IC1_ GFP/CHO cells. We found that the characteristic of adhesion was followed almost immediately (~10 min) by the rapid accumulation of ICAM-1 on CHO cells at a tight interface between the two cells. Interestingly, ICI_GFP/CHO cells projected plasma membrane and encircled approximately half surface of LFA-1+ cells, as defined by confocal microscopy. This unusual phenomenon was also confirmed on HUVEC after adhesion of LFA-1＊ cells. Neither cytochalasin D nor 2,3-butanedione 2-monoxime an inhibitor of myosin light chain kinase blocked LFA-1-mediated ICAM-1 clustering, indicating that actin cytoskeleton and myosin-dependent contractility are not necessary for ICAM-1 clustering. Taken together, we suggest that leukocyte adhesion to endothelial cells induces specialized form of ICAM-1 clustering that is distinct from immunological synapse mediated by T cell interaction with antigen presenting cells.