The techniques of IVM, IVF and IVC of canine oocytes may provide useful information for gamete salvage programs and the conservation of endangered canidae. This investigation has been made to determine the efficiency of in vitro maturation (IVM) as a basic experiment to study the development of canine oocytes after in vitro fertilization (IVF). The rate of oocytes developing to the MII stage was higher in the hormone treated group (10 IU/ml hCG+eCG, 14.7%, p<0.05) than in the control group (0 IU/ml hCG+eCG, 10.0%). The monospermy and pronuclear rates of canine oocytes were investigated after caffeine treatment on IVF. Canine oocytes were fertilized in the Fert‐TALP medium supplemented with 0, 10, 20 or 30 mM caffeine (Fert I, Fert II, Fert III or Fert IV, respectively). The highest pronuclear formation rate was obtained in the Fert I for 24 h IVF (6.7%, 6/89). Therefore, it is believed that unlike in other mammals, caffeine in canine IVF does not increase the efficiency of fertilization rate, and is not an important factor.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a naturally occuring polyphenol compound which present in the skin of grapes and red wine has been considered to posses chemopreventive and antioxidant properties. However, little is known about the cellular actions by which resveratrol mediates its therapeutic effects. In this study, the effect of resveratrol on cell proliferation and induction of apoptosis in human osteogenic sarcoma (HOS) cells was investigated. IC50 value was determined to be approximately 60μg/mℓ. Chromosomal DNA framgmentation analysis showed the appearance degraded DNA in time-and dose-dependent manner upon treatment of resveratrol. In order to observe the molecular mechanism involved in resveratrol-induced apoptosis, Western blot analysis was performed. We observed the decrease in the level of procaspase-3, the zymogen form of active caspase-3 in resveratrol-treated cells. This result implies that caspase-3 is activated upon treatment of resveratrol. The activation of caspase-3 was confirmed by the cleavage of poly(ADP-ribose) polymerase. Taken together, our data demonstrate that resveratrol has anti-proliferative effect on HOS cells and induced apoptosis through activation of caspase-3 and PARP cleavage.

This study aimed to produce fermented soy-powder milk (FSPM) with Lactobacillus plantarum P1201 and to evaluate its anti-obesity activity. Isoflavone and conjugated linoleic acid (CLA) of unfermented soy-powder milk (UFSPM) and FSPM and were analyzed via high-pressure liquid chromatography (HPLC) and gas chromatography (GC). Their inhibitory activities against α-glucosidase, α-amylase, and pancreatic lipase were assayed. Their anti-obesity activities were evaluated on the basis of their inhibitory effects on adipocyte differentiation in 3T3-L1 cells, and the expression of mRNAs associated with adipogenesis and lipid metabolism were analyzed via real time-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR). FSPM with L. plantarum P1201 increased the isoflavone aglycones (daidzein, glycitein, and genistein) content and produced CLA in soy-powder milk (SPM), both of which possessed bio-activity. Both UFSPM and FSPM showed dose-dependent inhibitory activity for α-glucosidase, α-amylase, and pancreatic lipase. FSPM, but not UFSPM, suppressed adipogenesis in 3T3-L1 cells and reduced their triglyceride content by 23.1% after treatment with 1,000 μg/mL of FSPM, compared with the control group. The anti-obesity effect of FSPM can be attributed to CLA and isoflavone aglycones, which targeted CCAAT/enhancer binding protein α (C/EBP-α) and down-regulated lipoprotein lipase (LPL), adiponectin, adipocyte fatty acid-binding protein (aP2), fatty acid synthase (FAS), and acetyl CoA carboxylase (ACC) mRNA. Furthermore, FSPM enhanced the inhibitory activity of glucosidase and pancreatic enzymes and anti-obesity activity. Further studies are required to investigate whether the anti-obesity effect of FSPM persists in an in vivo mouse model of diet-induced obesity.

Dendropanax morbifera Leveille (Araliaceae) is an endemic species growing in the south-western part of South Korea that has been used in folk medicine and health functional food. In this study, we investigated an extract of quercetin in Jeju D. morbifera by varying different parts (fruit, sprouts, leaves, sprigs, and branches), harvest times, and extraction solvents. In addition, we aimed to establish a simple and reliable HPLC/UV analytical method to determination of quercetin for the quality control and base line data of the Jeju D. morbifera extract as a health functional food ingredient. The analytical specificity was determined with retention time and photo diode array (PDA) spectrum by analyzing quercetin using HPLC and comparing the results to those of extracts. This analytical method for quercetin was validated for its limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. A high linearity in the standard calibration curve was obtained, with a coefficient of determination (R2) of 0.9996. Also, the LOD and LOQ values were found to be 0.28 μg/mL and 0.85 μg/mL, respectively, and the recoveries of quantified compounds ranged from 97.91% to 104.10%. Furthermore, the relative standard deviation (RSD) values of data from the intra- and inter-day precision analyses were less than 1.36% and 3.65%, respectively. As a result, the highest quercetin content among the extracts of Jeju D. morbifera leaves was found to be 20.14 mg/g, which was extracted at harvest in May (cultivation period 10 years) with 60% EtOH. All in all, we believe that the results obtained would be helpful in the development of nutraceutics and natural medicines and for the quality control of D. morbifera.

Background : Cynanchum wilfordii and Cynanchum auriculatum belong to the Asclepiadaceae family and appear morphologically similar. In order to discriminate them, it is needed to find the presence of sap and the leaf shapes: C. auriculatum has a blade ovate leaf comparing to C. wilfordii. However, in the herbal medicine market, they have been handled as cut and dried roots. Due to their similar morphology, it is limited to distinguish the roots of C. wilfordii and C. auriculatum. Recently in Korea, it has been a critical issue to misuse these two roots in the herbal market and food industry. Thus, it is required to establish a robust tool for the discrimination and quality control of them. Methods and Results : To separation and characterization of flavor compounds, C. wilfordii and C. auriculatum samples were analyzed by head space solid phase micro extraction (HSS) fiber coupled with gas chromatography-mass spectrometry using Rtx-5MS (30 m x 0.25 mm x 0.25 μm) column. As a result, We have identified compounds of a few hundred in aliphatic aldehydes and aliphatic alcohols, alkenes and acids, aromatic compounds, aromatic compounds containing nitrogen & sulfur, etc,. In particular, The aliphatic and aromatic compounds had been clearly separated on the second dimensional direction by using two-dimensional GC. Conclusion : The volatile flavor compounds of C. wilfordii and C. auriculatum could easily analyzed without pre-treatment with improved resolution and sensitivity using HSS-GCxGC-TOFMS. We have identified compounds of a few hundred in C. wilfordii and C. auriculatum sample. And It was more accurately qualitative confirmed with separation of GCxGC and TSD. We have confirmed the PCA and PLS-DA Plot that was classified depending on C. wilfordii and C. auriculatum through multivariate statistical analysis of the identified flavor compounds.

Background : Allergy is a common disease caused by type I hypersensitivity reaction of the immune system. Unfortunately, there is no proper treatment for allergy. Therefore, the discovery of therapeutic drugs for allergy is essential. In this study, the crude extracts of 56 plant parts were screened for anti-allergy effects in RBL-2H3 cells. Methods and Results : IgE-sensitized RBL-2H3 cells were individually treated with 56 extracts of medicinal herbs at the final concentration of 20 ㎍/㎖ and stimulated with the antigen DNP-BSA. β-Hexosaminidase release, interleukin-4 (IL-4) secretion, and cell viability in the sample treated cells were measured. Among the tested samples, extracts from the root of Polygonatum stenophyllum Maxim., aerial part of Acer triflorum Kom., and leaf of Pyrus pyrifolia var. culta (Makino) Nakai showed inhibitory effects on β-hexosaminidase release. The aerial part of Peucedanum japonicum Thunberg and seed of Panax ginseng C.A.Mey. showed suppressive activities on IL-4 secretion. All of the extracts were not cytotoxic at the tested concentration. Conclusion : From the result, six extracts including Polygonatum stenophyllum Maxim. (root) and Acer triflorum Kom. (aerial part) inhibited both β-hexosaminidase release and IL-4 secretion in IgE-sensitized RBL-2H3 cells. The use of these extracts for developing anti-allergy materials is suggested.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs

The patent ductus arteriosus (PDA) is a vascular structure connecting the proximal descending aorta to the roof of the main pulmonary artery, near the origin of the left branch pulmonary artery. Transcatheter closure has become the treatment of choice for most cases of PDA in both children and adults; however, measurement of the exact size and morphology of the shunt in adult cases using only contrast fluoroscopy is difficult. We report on a case of a 49-year-old woman who underwent transcatheter closure of PDA with intravascular ultrasound (IVUS) guideance. In the current case, IVUS is feasible and helpful for measuring the exact size and shape of the PDA.

Chronic gamma irradiation can be used an alternative mutation breeding methods for induction of many useful mutants. Seedlings of purple-colored wheat plants were irradiated with wide range doses of chronic gamma-rays (20, 25, 30, 40, 50, 70, 100, 125, 150, 200, 250, 300 Gy) during 6 weeks at gamma-phytotron in the Korea Atomic Energy Research Institute, respectively. To identify the biological responses purple-colored wheat, we examined the plant height, chlorophyll, carotenoid and total anthocyanin contents in leaf. Plant growth, chlorophyll and carotenoid contents in leaf were decreased when the dose rate increased. Anthocyanin contents were increased with the increase of the radiation dose until 50 Gy treatment. To confirm the real contents of anthocyanin, we also investigated cyanidin-3-glucoside in purple-colored wheat leaf by using UPLC analysis. These results indicate that anthocyanin accumuation was observed under chronic gamma irradiation.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Kaposi's sarcoma is a lympho-angioproliferative disease that primarily affects the skin and subcutaneous tissues. Most importantly, Kaposi’s sarcoma occurs in the immunocompromised, especially AIDS patients and organ transplant recipients being treated with immunosuppressants. The occurrence of classic Kaposi’s sarcoma after the diagnosis of any primary solid tumor has rarely been reported. We experienced a 70-year-old man, who had received surgery and chemoradiotherapy for the treatment of primary gallbladder cancer, that developed multiple tender deep purple papules on his trunk, back and feet which were proven to be pathologic symptoms of Kaposi’s sarcoma.