• Korean Journal of Pharmacognosy
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• v.40 no.3
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• pp.196-204
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• 2009

This study was carried out to investigate the in vivo and in vitro inhibitory effect of a traditional herbal complex (HC) extract prepared from a mixture of four oriental herbs (Dioscorea Rhizoma, Glycine soja Sieb. et Zucc, Bombycis corpus, Fermented Glycine soja) that have been widely used for the treatment and prevention of diabetes mellitus on hyperglycemia. The water extract of HC showed potent inhibitory effect on $\alpha$-glucosidase with $IC_{50}$ value of 1.24 mg/mL. Additionally, the ethanol extract of HC was also found to exhibit significant inhibitory effect against protein tyrosine phosphatase $1{\beta}$ ($PTP1{\beta}$), which is known as a major regulator of both insulin and leptin signaling. In the $PTP1{\beta}$ inhibitory assay, the most active n-hexane fraction obtained from the ethanol extract of HC, was identified as a mixture of fatty acid derivatives by gas chromatography-mass spectrometry (GC-MS). In high-fat diet-low dose streptozotocin (STZ)-induced diabetic rat, the water extract of HC improved the oral glucose intolerance as compared with rosiglitazone. HC also caused a marked decrease of body weight and fasting blood glucose and a significant improvement on glucose tolerance in metabolic syndrome mice model. These findings support that this traditional HC may be useful in the control of blood glucose in diabetes mellitus and metabolic syndrome.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.3
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• pp.470-486
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• 1995

Mammary epithelial cells contain a subpopulation of cells with a large proliferativ potential which are responsible for the maintenance of glandular cellularity and are the progenitor cells of mammary cancer. These clonogens give rise to multicellular clonal alveolar or ductal units(AU or DU) on transplantation and hormonal stimulation. To isolate putative mammary clonogens, enzymatically monodispersed rat mammary epithelial cells from organoid cultures and from intact glands are sorted by flow cytometry according to their affinity for FITC labeled peanut lectin(PNA) and PE labeled anti-Thy-1.1 antibody(Thy-1.1) into four subpopulations : cells negative to both PNA and Thy-1.1(B-), PNA+cells, Thy-1.1+cells, and cells positive to both reagents(B+). The in vivo transplantation assays indicate that the clonogenic fractions of PNA+cells from out-growths of organoids in primary cultures for three days in complete hormone medium(CHM) are significantly higher than those of cells from other subpopulations derived from cultrues or from intact glands. Extracellular matrix(ECM) is a complex of several proteins that regulated cell function ; its role in cell growth and differentiation and tissue-specific gene expression. It can act as a positive as well as a negative regulator of cellular differentiation depending on the cell type and the genes studied. Regulation by ECM is closely interrelated with the action of other regulators of cellular function, such as growth factors and hormones. Matrigel supports the growth and development of several different multicellular colonies from mammary organoids and from monodispersed epithelial cells in culture. Several types of colonies are observed including stellate colonies, duct-like structures, two- and three-dimensional web structures, squamous organoids, and lobulo-duct colonies. Organoids have the greatest proliferative potential and formation of multi-cellular structures. Phase contrast micrographs demonstrate extensive intracellular lipid accumulation within the web structures and some of duct-like colonies. At the immunocytochemical and electron micrograph level, casein proteins are predominantly localized near the apical surface of the cells or in the lumen of duct-like or lobulo-duct colonies. Squamous colonies are comprised of several layers of squamous epithelium surrounding keratin pearls as is typical fo squamous metaplasia(SM). All-trans retinoic acid(RA) inhibits the growth of SM. The frequency of lobulo-ductal colony formation increased with the augmentation of RA concentration in these culture conditions. The current study models could provide powerful tools not only for understanding cell growth and differentiation of epithelial cells, but also for the isolation and characterization of mammary clonogenic stem cells.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.235-243
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• 2002

This study was designed to examine the ability of the bovine (MII) oocytes cytoplasm to support several mitotic cell cycles under the direction of differentiated somatic cell nuclei of bovine, porcine, mouse and human. Bovine GV oocytes were matured in TCM-199 supplemented with 10% FBS. At 20h after IVM, recipient oocytes were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst and their 1st polar body (PB) and MII plate were removed by enucleation micropipette under UV filter. Ear skin samples were obtained by biopsy from an adult bovine, porcine, mouse and human and cultured in 10% FBS added DMEM. Individual fibroblast was anlaysed chromosome number to confirm the specificity of species. Nuclear transferred (NT) units were produced by electrofusion of enucleated bovine oocytes with individual fibroblast. The reconstructed embryos were activated in 5 $\mu$M ionomycin for 5 min followed by 1.9 mM 6-dimethylaminopurine (DMAP) in CR1aa for 3 h. And cleaved NT embryos were cultured in CR1aa medium containing 10% FBS on monolayer of bovine cumulus cell for 8 days. Also NT embryo of 4~8 cell stage was analysed chromosome number to confirm the origin of nuclear transferred somatic cell. The rates of fusion between bovine recipient oocytes and bovine, porcine, mouse and human somatic cells were 70.2%, 70.2%, 72.4% and 63.0%, respectively. Also, their cleavage rates were 60.6%, 63.7%, 54.1% and 62.7%, respectively, there were no differences among them. in vitro development rates into morula and blastocyst were 17.5% and 4.3% in NT embryos from bovine and human fibroblasts, respectively. But NT embryos from porcine and mouse fibroblasts were blocked at 16~32-cell stage. The chromosome number in NT embryos from individual fibroblast was the same as chromosome number of individual species. These results show that bovine MII oocytes cytoplasm has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA.

• Development and Reproduction
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• v.6 no.2
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• pp.105-110
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• 2002

Numerous hormones are involved in the regulation of reproduction. Among them, estrogen and progesterone are the most important ovarian steroid hormones regulating female fertility. On the other hand, diverse stressors impede female receptivity and fertility. Since norepinephrine(NE) and epinephrine(E) are released from the adrenal during stress, it might play a role in stress-induced disruptions of fEmale reproductive parameters. The present study was performed to analyze the changes in adrenal catecholaminergic activities in cycling rats. The tissue content and secretion level of catecholamines were determined by high performance liquid chromatography coupled with electrochemical detector(HPLC-ECD). Adrenomedullary content of norepinephrine(NE) was increased on proestrus stage (59.47 $\pm$ 6.86 ug/gland), peaked on diestrus I stage(65.22 $\pm$ 5.99 ug/gland), and was nadir on diestrus II stage(41.63 $\pm$ 1.33 ug/gland). The highest E content was observed on proestrus stage(361.86 $\pm$ 15.58 ug/gland) while the lowest level was on diestrus II stage(285.58 $\pm$ 12.25 ug/gland). In addition to these observations, a significant reduction of the NE : E ratio was observed (1 : 4.81 on diestrus I vs 1 : 6.13～7.02 on other stages). In vitro secretion of adrenal NE and E was increased on proestrus stage, peaked on estrus stage, and decreased on diestrus II stage. Interestingly, the NE : E ratio in conditioned media was significantly increased on estrus stage (1 : 3.32 vs 1 : 2.34～2.65 on other stages. The biosynthesis of NE and E is mediated by tyrosine hydroxylase(TH) and phenylethanolamine-N-methyltransferase(PNMT) which acts conversion of tyrosine into DOPA and NE into E, respectively. These finding demonstrated that sex steroids, during setrous cycle, seem to be able to modify the adrenal catecholamines biosynthesis and secretion with stage-specific manner by modulation of the enzyme activities.

• Korean Journal of Medicinal Crop Science
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• v.16 no.5
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• pp.356-370
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• 2008

This study was conducted for screening on antioxidant activity of 429 plants and selecting new potential antioxidant candidates. In vitro test models such as scavenging activity on DPPH radical and inhibitory activity on linoleic acid oxidation were used in the preliminary study. Flower of Sanguisorba officinalis, flower of Sedum kamtschaticum, flower of Rumex obtusifolius, and root of Sedum kamtschaticum showed very effective antioxidant activity on DPPH radical and linoleic acid oxidation. Those plants showed 8.1, 9.4, 9.9, $11{\mu}g/ml$ in DPPH radical scavenging activity as $SC_{50}$ and did 80.4, 80.1, 84.5, 88.0% in inhibition activity on linoleic acid oxidation, respectively. Root of Sedum middendorfianum M. showed positive effects in superoxide radical scavenging activity ($38.4{\mu}g/ml$) and inhibitory effect on $CuSO_4$-induced LDL oxidation (53.8% at final concentration of $1{\mu}g/ml$). Gleditsia japonica Mig. showed high antioxidant activity on LDL oxidation as 71.6% at final concentration of $1{\mu}g/ml$ and total phenol content of 958.5 mg% as tannic acid equivalent. In conclusion, we think that these plants having potent antioxidant activity might be studied further and could be used as new resources for many purposes including healthy food, functional cosmetics and drug development etc.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Journal of Life Science
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• v.23 no.9
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• pp.1079-1087
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• 2013

Speed germination success and robust vegetative growth of citrus rootstock through improved sowing methods and fertilizer inputs offer the usage of root system for the citrus. The current study evaluated the influence of seed coat removal and different fertilizer concentrations on plant germination and plant growth of spontaneous rootstock siblings. Decoated and coated seeds of diploid and tetraploid plants were sown in tubes. Commercial fertilizer concentrations of 0, 2, 4, 6, 8 and $10g{\cdot}l^{-1}$ were added. The experimental layout followed a randomized block $2{\times}6$ factorial design (seed coat removal ${\times}$ fertilizer concentration) for each rootstock. Fertilizer concentrations were 0, 10, 20 and $30g{\cdot}l^{-1}$ of the fertilizer for the resistance of the strength on the salt level. The germination rate of seeds without testa sown in vitro was improved (67-80%) compared to that of nontreated seeds. The eventual tree height of the seeds without testa in the diploid group was increased due to higher fertilization compared to that in the nontreated group. The removal of seed testa promoted the seed germination of both diploid and tetraploid trifoliate orange and resulted in greater height. Their vegetative development was also increased due to the increased fertilization of the rootstock. The Fv/Fm value for the diploid plants was 0.4 and 0.8 for the tetraploid ones under salt stress after 11 days of treatment. The removal of seed testa may improve the seed germination of trifoliate orange. Tetraploid trifoliate orange appears to possess resistance to salt stress compared to the diploid variety.

• Journal of the korean academy of Pediatric Dentistry
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• v.27 no.1
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• pp.169-179
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• 2000

The purpose of this study is to develop the system which convert the optical difference of teeth texture between intact enamel and incipient caries lesion into shade difference by laser fluorescence and to develop new and simple caries activity test using laser fluorescence. The experimental design of this study consists of three parts. In first part, a new method for the in vitro assessment of changes in initial enamel caries lesion of Bovine teeth using laser fluorescence is tested. In second part, in vivo assessment undertaken. Number of teeth which showed incipient carious lesion on buccal surface examined by laser fluorescence was compared with the caries activity test of $Cariescreen^{(R)}$ test and other oral environmental test of dDfFtT. In third part, new caries activity test measured by laser fluorescence was developed on the basis of above results and evaluated the sensitivity, specificity, and diagnostic power. Optical density measured by laser fluorescence was increased as increasing the depth of incipient carious lesion and showed high correlation$(\gamma=0.7015)$ with lesion depth. Optical density showed direct proportion to lesion depth. Linear equation was obtained between the optical density and the lesion depth by regression analysis. The result of caries activity test with laser fluorescence showed high correlation with those of $Cariescreen^{(R)}$ test and dDfFtT examination. Caries activity test with laser fluorescence showed 48% of sensitivity, 52% of specificity, and 45% of diagnostic power on the basis of dDfFtT examination, and also showed 48% of sensitivity, 51% of specificity, and 36% of diagnostic power on the basis of $Cariescreen^{(R)}$ test. In regard above result, caries activity test with laser fluorescence considered to be reliable for caries activity test compared with other oral environmental test. and it was also considered to be practical because it would be simple, inexpensive, and time saving method.

• Development and Reproduction
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• v.6 no.1
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• pp.55-66
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• 2002

Embryonic stem cells(ES cells) are derived from the inner cell mass(ICM) of blastocysts, which have the potentials to remain undifferentiated, to proliferate indefinitely in vitro, to differentiate into the derivates of three embryonic germ layers. ES cells are an attractive model system for studying the initial developmental decisions and their molecular mechanisms during embryogenesis. Additionally, ES cells of significant interest to those characterizing the various gene functions utilizing transgenic and gene targeting techniques. We investigated the effects of reproductive hormones, gonadotropins(GTH) and steroids on the induction of differentiation and expressions of their receptor genes using the newly established mouse ES cells. We collected the matured blastocysts of inbred mice C57BL/6J after superovulation and co-cultured with mitotically inactivated STO feeder cells. After 5 passages, we confirmed the expression alkaline phosphatase(Alk P) activity and SSEA-1, 3, 4 expressions. The protocol devised for inducing ES differentiation consisted of an aggregation steps, after 5 days as EBs in hormone treatments(FSH, LH, E$_2$, P$_4$, T) that allows complex signaling to occur between the cells and a dissociation step, induced differentiation through attachment culture during 7 days in hormone treatments. Hormone receptors were not increased in dose-dependent manner. All hormone receptors in ES cells treated reproductive hormones were expressed lower than those of undifferentiated ES cell except for LHR expression in E$_2$-treated ES cells group. After hormone induced differentiation, at least some of the cells are not terminally differentiated, as is evident from the expression of Oct-4, a marker of undifferentiated. To assess their differentiation by gene expression, we analyzed the expression of 7 tissue-specific markers from all three germ layers. Most of hormone-treated group increased in the expression of gata-4 and $\alpha$ -fetoprotein, suggesting reproductive hormone allowed or induced differentiation of endoderm.

• Development and Reproduction
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• v.5 no.1
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• pp.47-52
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• 2001

Predetermination of sex in livestock of offpring is in great demand and is of critical importance to providing for the most efficient production of the animal ariculture. Such a sexing techlology would also enhance the economy of conventional artificial insemination(AI) and aid the porcine industry. The purpose of this study was to evaluate the efficiency of enriching X-bearing porcine sperm using discontinuous percoll gradients and PCR mefhod. Semen was collected from mature boars of proven fertility center (AI center KimHae). Sperm was leaded on the isotonic discontinuous percoll gradient and then it was centrifuged at 120 ${\times}$ g for 20 minutes. After centrifugation, sperm included in each fraction were recovered (7${\times}$10$^6$ sperms/ml) and then sperm genomic DNA was extractedfor the PCR. SRY gene was used to evaluate the ratio between X and Y sperm in the separated fractions. Ju viro ffrtilization wascarried out by adding the unseparated sperm (control) or separated (experimental poop) to the matured oocytes in TCM-199. Embryos for sex determination were obtained at 2 cell stage and then was used for SRY gene amplification. After centrifugation of discontinuous percoll gradient, the most motile sperm was obtained at 95% fiaction (94.4% ${\pm}$ 5.1%, p < 0.01). The PCR analysis evaluated that 30%, 50% and 65% fractions were Y sperm rich, whereas 80% and 95% fractions were X sperm rich. PCR analysis with each porcineembryo showed that 33.3% of control and 66.7% of experimental group were determined as female embryos. In conclusion, in vitro matured oocytes inseminated with sperms (95% fraction) prepared by percoll gradient centrifugation showed high fertilization rates and female embryos than control sperms.