• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.126-126
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• 1998

Studies on natural products are of great interest, due to the limits in development of synthesized medicine and its side effects. Deer antler is the most popular cure-all type drug among Asian folk medicines. In this study, we newly isolated the biologically active components from chloroform extract and 70% ethanol extract of deer antler, and analyzed their structures. First, the structure of monoacetyldiglyceride in deer antler was identified. To investigate the structure-activity relationship of monoacetyldiglycerides, we synthesized diverse substituted glycerides from glycerol, and confirmed their structures by spectroscopic methods. Among seven structurally-interesting compounds tested in this study, compound 1,2,3,5, and 6 showed activity toward [Ca$\^$2+/]$\_$i/ increase in fura-2 loaded rat pancreatic acinar cells. Second, 70% ethanol extract of deer antler stimulated insulin release from rat pancreatic islets. We found the most effective fraction was CN-Es-8 in 70% ethanol extract, and it increased intracellular Ca$\^$2+/ concentration in pancreatic ${\beta}$-cell.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.335-341
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• 1997

The aims of this study are to establish a stable isolation method of blastomeres from bovine early embryos and examine their developmental potential in vitro Early embryos were produced by maturation and fertilizaion in vitro of bovine follicular oocytes. Blastomeres were isolated from 2~8-cell embryos in $Ca^2$+-, $Mg^2$+-free PBS+EDTA after removing the zonae pellucidae Isolated blastomeres were cultured in CZB containing BOEC for upto 240 hpi. Cleavage rates of them were 18.5%(10 /54) in 1 /2 blastomeres, 33.3%(16/48) in 1/4 blastomeres and 34.2%(14 /41) in 1/8 blastomeres, respectively. The rates of blastocystic vesicle formed were 8.7%(4 /46) in 1/2 blastomeres, 26.6% (17/64) in 1/4 blastomeres and 10.3%(8 /78) in 1/8 blastomeres, respectively. Blastomeres developed into various types of blastocystic vesicles and trophoblastic vesicles as evidenced by the Hoechst 33258 staining and morphology. This results suggest that the isolation method used and subsequent culture of isolated blastomeres from bovine early embryos should be useful to obtain extra embryonic cells for various analyses such as PCR and putative ES cell culture.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.33-33
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• 2003

This study was conduct to compare the efficacy to produce male and female somatic cloned piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1mM dibutyryl cyclic adenosine monophosphate (dbc-AMP, Sigma, USA), and 0.1 IU/ml human menopausal gonadotrophin (hMG, Teikokuzoki, Japan) for 20h and then cultured without dbcAMP and hMG for another 18 to 24 h. Female and male fetal cells were isolated from each fetus, cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/ml BSA under mineral oil at 39$^{\circ}C$ in 5% $CO_2$ in air. A total of 12,328 nuclear-transferred embryos (1- to 4-cell stage) were surgically transferred into 69 surrogate gilts. Three recipients aborted during the period of conception. Three gilts delivered eleven female piglets, and five recipients gave rise to birth 22 male piglets. The average birth weigh of the cloned piglets was 1.52 kg (1.38~1.83 kg) in female piglets and 0.84 kg (0.45~1.25 kg) in male piglets. Alive cloned pigs was seven in female piglets (63.6%) and four in male piglets (18.2%). The other two recipients is ongoing. This study suggests that female-derived fetal cell as a nuclear donor has more capability on production of cloned piglets than male.

• Korean Journal of Pharmacognosy
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• v.31 no.3
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• pp.273-279
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• 2000

Leukotriene $B_4\;(LTB_4)$ is a pro-inflammatory mediator synthesized in myeloid cells from arachidonic acid. Elevated levels of $LTB_4$ have been found in a number of inflammatory diseases and levels are related to disease activity in some of these. Because $LTB_4$ interacts with cells through specific cell surface receptors, $LTB_4$ receptor blockade is the most specific approach to reduce the pathogenic role of $LTB_4$. In order to find $LTB_4$ receptor antagonist from plants, we screened the $LTB_4$ receptor antagonistic activity of the methanol extract and solvent fractions of herbal drugs. The ability of samples to inhibit specific binding of $[^3H]-LTB_4$ to human peripheral neutrophils was used as assay to evaluate the antagonistic activity of plant materials. Among the tested methanol extracts of herbal drugs, Mori Radicis Cortex, Perillae Semen, Armeniacae Semen and Sophorae subprostratae Radix showed potent inhibitory activity above 70% at the concentration of $100\;{mu}g/ml$. The inhibitory activities of $LTB_4$ binding to human neutrophils were evaluated for several solvent fractions at three different concentrations. Especially, hexane soluble fractions of Anemarrhenae Rhizoma and Embeliae Radix, and ethyl acetate soluble fractions of Aristolochiae Fructus, Magnoliae Cortex and Zingiberis Rhizoma crudus showed moderate activity at $25\;{mu}g/ml$. These fractions were promising candidates for the study of the activity-guided chromatographic purification of active compounds. Silica gel column chromatography of hexane soluble fractions of Anemarrhenae Rhizoma and Embeliae Radix gave very active sub-fractions, AA-4 and ES-4, and their inhibition activities of $LTB_4$ binding to human neutrophil at $30\;{mu}g/ml$ were 78% and 62%, respectively. From these results we could anticipate new $LTB_4$ receptor antagonist from herbal drugs, and the block of $LTB_4$ effects may provide beneficial in neutrophil mediated diseases such as inflammation and bronchial asthma.

• Journal of Life Science
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• v.17 no.4 s.84
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• pp.587-592
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• 2007

Manipulation of the mouse genome by activating or inactivating the gene has contributed to the understanding of the function of the gene in the subset of cells during embryonic development or postnatal period of life. Most of all, gene targeting, which largely depends on the availability of mouse embryonic stem (ES) cells, is the milestone of development of animal models for human disease. Recombinase-mediated genome modification (Cre-LoxP and Flp-Frt etc) and the ligand-dependent regulation system, more accurate and elaborate manipulation tools, have been successfully developed and applied to dissect the mechanisms governing complex biological processes and to understand the role of protein in temporal-and spatial aspects of development. As technologies concerning refined manipulation of mouse genome are developed, they are expected to open new opportunities to better understand the diverse in vivo functions of genes.

• Physical Therapy Korea
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• v.14 no.1
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• pp.28-36
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• 2007

The aim of this study was to identify the effect of suspension unloading (SU) and electrical stimulation upon the development of neonatal muscular system. For this study, the neonatal rats were randomly divided into three groups: a control group, an experimental group I, and an experimental group II. The SU for experimental group I and II was applied from postnatal day (PD) 5 to PD 30. The electrical stimulation for soleus muscle of experimental group IIwas applied from PD 16 to PD 30 using neuromuscular electrical stimulation (NMES), which gave isometric contraction with 10 pps for 30 minutes twice a day. In order to observe the effect of SU and ES, this study observed myocyte enhancer factor 2C (MEF2C) and vascular endothelial growth factor (VEGF) immunoreactivity in the soleus muscles at PD 15 and PD 30. In addition, the motor behavior test was performed through footprint analysis at PD 30. The following is the result. At PD 15, the soleus muscles of experimental group Iand II had significantly lower MEF2C, VEGF immunoreactivity than the control group. It proved that microgravity conditions restricted the development of the skeletal muscle cells at PD 15. At PD 30, soleus muscles of the control group and experimental group II had significantly higher MEF2C, VEGF, immunoreactivity than experimental group I. It proved that the NMES facilitated the development of the skeletal muscle cells. At PD 30, it showed that SU caused the decrease in stride length of parameter of gait analysis and an increase in toe-out angle, and that the NMES decreased these variations. These results suggest that weight bearing during neonatal developmental period is essential for muscular development. They also reveal that NMES can encourage the development of muscular systems by fully supplementing the effect of weight bearing, which is an essential factor in the neonatal developmental process.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Molecules and Cells
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• v.26 no.4
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• pp.338-343
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• 2008

An embryonic stem cell is a powerful tool for investigation of early development in vitro. The study of embryonic stem cell mediated neuronal differentiation allows for improved understanding of the mechanisms involved in embryonic neuronal development. We investigated expression profile changes using time course cDNA microarray to identify clues for the signaling network of neuronal differentiation. For the short time course microarray data, pattern analysis based on the quadratic regression method is an effective approach for identification and classification of a variety of expressed genes that have biological relevance. We studied the expression patterns, at each of 5 stages, after neuronal induction at the mRNA level of embryonic stem cells using the quadratic regression method for pattern analysis. As a result, a total of 316 genes (3.1%) including 166 (1.7%) informative genes in 8 possible expression patterns were identified by pattern analysis. Among the selected genes associated with neurological system, all three genes showing linearly increasing pattern over time, and one gene showing decreasing pattern over time, were verified by RT-PCR. Therefore, an increase in gene expression over time, in a linear pattern, may be associated with embryonic development. The genes: Tcfap2c, Ttr, Wnt3a, Btg2 and Foxk1 detected by pattern analysis, and verified by RT-PCR simultaneously, may be candidate markers associated with the development of the nervous system. Our study shows that pattern analysis, using the quadratic regression method, is very useful for investigation of time course cDNA microarray data. The pattern analysis used in this study has biological significance for the study of embryonic stem cells.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.166-175
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• 2018

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line ($Rag-2^{-/-}$) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. $Rag-2^{-/-}$ mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in $Rag-2^{-/-}$ mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in $Rag-2^{-/-}$ mice. These findings indicate that our $Rag-2^{-/-}$ mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.28 no.4
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• pp.74-91
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• 2015

Background and Objective : Asthma is a chronic inflammatory disease at the mucosa and is associated with excess production of Th2 cytokine and eosinophil accumulation in lung.Gamchomahwang-tangextract(GME) is one of the well known prescription used in oriental medicine for treating asthma. This study was designed to compare the anti-asthmatic effect of GME according to the ratio of 2 compounds.Methods : To examine the effects of GME on asthma, mice were sensitized with 100 ㎍ of OVA and 1 ㎎ of aluminum potassium sulfate(Alum; Sigma) intraperitoneally on day 1 and 15. From day 22, mice were challenged on 3 consecutive days with 5% OVA. The anti-asthmatic effects of GME were evaluated by enhanced pause(Penh), bronchoalveolar lavage fluids (BALF), inflammatory cytokine production and genes expression, serum IgE production. and histological change in lung tissue. GMEⅠ consists of ES and GU in the proportion 2:1(300 ㎎/㎏ group), GMEⅡ consist of ES and GU in the proprtion 4:1(300 ㎎/㎏ group).Results : GMEⅠ,Ⅱ generally inhibited lung inflammation, inflammatory cells infiltration and cytokine production and gene expression such as IL-4, IL-5 and IL-13 in BALF and serum IgE level. GMEⅡ significantly reduced the cytokine production and gene expression such as IL-4, IL-5 and IL-13 in BALF and GMEⅠ decreased cytokine production of IL-4, IL-13 in BALF and gene expression of IL-4, IL-5 in Lung. GMEⅡ potently inhibited the development of Penh and also reduced the number of eosinophil during OVA-induced AHR(airway hyper-reactivity). Overall the results show that GMEⅡ has more effect on inhibiting production, gene expression of cytokine, serum IgE level and development of Penh than GMEⅠ. Consequently, GMEⅡ might be more effective than GMEⅠ at inhibiting allergic asthma on the OVA-induced mice model.Conclusion : These results indicate that GME has a deep inhibitory effects on airway inflammation and hyperresponsiveness in mice model of asthma and that suppression of IL-4, IL-5, IL-13 expression and decrease of IL-4, IL-5, IL-13 production in BALF might contribute this effect. Hence, the results indicate that GME might be useful herbal medicine of allergic asthma. As a result, GMEⅡ mght be superior to GMEⅠ in the aspect of anti-asthmatic effect on the OVA-induced mice model.