EGCG has inhibitory effect on a variety of cancers by inducing apoptosis and cell cycle arrest or inhibiting angiogenesis and metastasis. EGCG has been found to induce apoptosis in salivary gland carcinoma cells. But the potential anti-invasive effect of EGCG in salivary gland cancer has not been studied yet. The aim of this study is to evaluate the effect of EGCG on salivary gland adenocarcinoma SGT cell adhesion and migration to Type I collagen treatment. Western blot, adhesion and migration assay were performed to evaluate the impacts of EGCG on the expression of MMP-2/-9 and its upstream signaling molecules after treatment of type I collagen. SGT cell adhesion to type I collagen is significantly suppressed by EGCG. EGCG decreased expression of β1 integrin, phosphorylation of FAK, MMP-2/-9 compared with type I collagen treatment. In addition, EGCG inhibited the migration of SGT cells treated with type I collagen. These results suggest that EGCG could effectively inhibit the invasion and migration of human SGT cells by downregulating the expression of β1 integrin and MMP-2/-9 and phosphorylation of FAK, Akt, and Erk. Adhesion and migration to type I collagen of SGT cells can be influenced through EGCG.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass cells of blastocyst with the potential to maintain an undifferentiated state indefinitely. Fully characterized ES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, ES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various diseases in animals as well as in humans. However, important problems associated with ES cells from in vitro fertilized blastocysts particularly from humans must be resolved before taking up its therapeutic applications. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. This review therefore focuses on ES cells with respect to in vitro propagation and differentiation in basic cell and developmental biology for successful use of these cells in therapeutics.

The central granular cell odontogenic tumor is a rare benign odontogenic neoplasm of uncertain hisotogenesis characterized by varying amounts of eosinophilic granular cells and apparently inactive odontogenic epithelium with variable presence of calcified tissue. We present a case of central granular cell odontogenic tumor involving the maxilla of 35-year-old man with immunohistochemical characterization of granular cells. In microscopic view, the granular cells densely packed in sheets and lobules with abundant eosinophilic, granular cytoplasm and eccentric round-to-ovoid nuclei revealed immunoreactivity for vimentin, α1-antitrysin and CD68, and NSE but not for cytokeratin and S-100 protein while the interspersed odontogenic epithelial cells were positive for cytokeratin only. Granular cells also revealed strong PAS staining. Numerous concentric structured round to ovoid calcified aggregates were also noted. The lesion was treated with excision without recurrence for 8 years. Our immuohistochemical staining findings also suggest that the granular cells of central granular cell odontogenic tumor are mesenchymal in origin with possible histiocytic differentiation

Central granular cell odontogenic tumor (CGCOT) is a rare benign odontogenic neoplasm, with approximately 30 cases having been reported. The pathogenesis of CGCOT as well as the designation of this lesion is controversial because of unknown histogenesis of the granular cell. We present an additional case of CGCOT involving the mandible of a 50-year-old Korean man who complained of asymptomatic swelling of the right buccal gingiva. Current lesion is microscopically characterized by densely packed polyhedral granular cells surrounding interspersed islands or strands of odontogenic epithelium. Immunohistochemically, granular cells were positive for Vimentin and CD68, and negative for cytokeratin and S-100. These features support a mesenchymal origin for the granular cells as other results previously reported.

The purpose of this study was to investigate the effect of cytokine-induced osteoclastogenesis on tooth movement related to orthodontic force. We evaluated the cytotoxicity as well as the expression of OPG and RANKL, which influence the homeostasis of bone metabolism. Titanium particles were applied to human periodontal ligament cells and subcultured fourth generation cells. The ALP assay and the MTT assay were used to assess changes in cytotoxicity. After 48 hours, cytotoxicity increased proportionally with the concentration of titanium. With 20 mg, the cytotoxicity was the lowest. R T-PCR was u sed for assessing m R NA l evels of O PG a nd R ANKL; after 96 hours, t he m R NAs of O PG a nd R ANKL increased steeply. A western blot analysis showed that with 20 mg of titanium, the protein expression of OPG increased linearly with time, especially a fter 96 hours, while t he p rotein e xpression o f RANKL d id n ot s how significant changes with titanium processing. Given the increase in OPG expression after the initial cytotoxicity, changes in cytotoxicity with titanium may be attributable to the antagonistic effect of OPG on cytotoxicity

The present study aimed to verify the effects of DFO on PDL cells, with particular emphasis on focusing on osteoblastic differentiation. Its mechanisms related to heme oxygenase-1 (HO-1) pathway were also analyzed. DFO increased the expression of HO-1 and early osteoblastic differentiation markers, such as alkaline phosphatase (ALP) and bone sialoprotein (BSP). DFO upregulated heme oxygenase-1. Treatment with HO-1 siRNA blocked the DFO-stimulated osteoblastic differentiation and HO-1 expression. The NF-kB inhibitor pyrrolidine dithiocarbamate, phosphatidylinositol 3-kinase inhibitor Wortmannin, and p38 MAPK inhibitor U0126 blocked the effects of DFO on HO-1 expression and osteoblastic differentiation in PDL cells. Collectively, these data suggest that DFO promotes osteoblastic differentiation and induces the expression of defense protein HO-1 probably via PI3K, p38 MAPK, and NF-kB signalling pathways in PDL cells.

Previously, we reported that the osmolarity conditions in the satellite region were affected CpG DNA methylation status while Pre-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. This study was conducted to investigate the DNA methylation status of repeat sequences in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaCl or 0.05 M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. The DNA methylation status of the Pre-1 sequences in blastocysts was characterized using a bisulfite-sequencing method. Intriguingly, in the present study, we found the unique DNA methylation at several non-CpG sequences at the Pre-1 sequences in all groups. The non-CpG methylation was hypermethylated in all three groups, including in vivo group (86.90% of PZM- 3; 83.87% of NaCl; 84.82% of sucrose; 90.94% of in vivo embryos). To determine whether certain non-CpG methylated sites were preferentially methylated, we also investigated the methylation degree of CpA, CpT and CpC. Excepting in vivo group, preference of methylation was CpT>CpC>CpA in all three groups investigated. These results indicate that DNA methylation of Pre-1 sequences was hypermethylated in CpG as well as non-CpG site, regardless modification of osmolarity in a culture media.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

A total of 222 udder-half milk samples of lactating goats were collected from two herds in Korea during 2008 and all samples were subjected to bacteriological examination. Somatic cell counts (SCC) were also determined for all samples except for 13 (5.9%), which were collected from halves of udders with clinical mastitis. A total of 85 bacteria were isolated from 82 (36.9%) of 222 milk samples tested. Staphylococci were the predominant pathogens, accounting for almost 70% of the isolates: Coagulase negative staphylococci (CNS) and S. aureus constituted 55% (47/85) and 14.1% (12/85), respectively. Among 209 samples tested for SCC, bacteria were isolated from 36 of 115 (31.3%) samples with SCC of <1×106 cells/㎖ and 38 of 94 (40.4%) samples that had SCC of ≥1×106 cells/㎖, respectively. All S. aureus were detected from samples with SCC of ≥1×106 cells/㎖, while 25 of 47 (61.0%) CNS were isolated from milk samples with SCC of <1×106 cells/㎖. Mean SCC of milk samples that harbored S. aureus and CNS was 4,787×103 cells/㎖ and >1×106 cells/㎖, respectively. All S. aureus and CNS isolates were susceptible to all antimicrobials tested except for penicillin, to which 2 (16.6%) S. aureus and 12 (25.5%) CNS isolates showed resistance.

Vanadium, a dietary micronutrient, has been reported to present interesting biological and pharmacological properties, including superoxide and nitric oxide scavenging effects. Low-dose ionizing radiation (LDR) is known to damage DNA and cause apoptosis of peripheral immunocytes by producing reactive oxygen species (ROS). The aim of this study was to elucidate the capacity of immune activation of Jeju water containing vanadium on immunosuppression caused by LDR. We examined the ROS production, DNA damage, cell apoptosis and proliferation of peripheral immunocytes in irradiated mice drinking different concentrations for 90 days; V0 (vanadium 0㎍/L, control), V1 (vanadium 15～20㎍/ L) and V2 (vanadium 20∼25㎍/L). Compared to V0 control where level of ROS showed tendency to increase, the ROS production was attenuated in peripheral immunocytes of irradiated mice drinking V1 and V2. DNA damage of peripheral immunocytes triggered by LDR significantly increased in mice drinking V0 compared to non-irradiated control, whereas V1 and V2 dramatically induced remission of DNA damage. On the observation of apoptosis of peripheral immunocytes, V1 and V2 showed the potency to reduce the number of apoptotic cells. On the other hand irradiated mice drinking V0 exhibited raised number of apoptotic cells. From the results obtained, we speculated that Jeju water containing vanadium (V1 and V2) has a potential role in decreasing DNA damage and apoptosis of immune cell by inhibiting ROS production. Consistent with this, Jeju water containing vanadium (V1 and V2) exhibits a capacity to enhance cell proliferation of peripheral immunocytes, which is suppressed by LDR as shown in V0 control. Collectively, Jeju water containing vanadium reduced DNA damage and apoptosis and induced the stimulatory potential on immunocytes. These results suggest that Jeju water containing vanadium sustained immune activities under immunosuppression caused by LDR.

The functional cardiovascular system is comprised of distinct mesoderm-derived lineages including endothelial cells, vascular smooth muscle cells and other mesenchymal cells. Recent studies in the human embryonic stem cell differentiation model have provided evidence indicating that these cell lineages are developed from the common progenitors such as hemangioblasts and cardiovascular progenitor cells. Also, the studies have suggested that these progenitors have a common primordial progenitor, which expresses KDR (human Flk-1, also known as VEGFR2, CD309). We demonstrate here that sustained activation of BMP4 (bone morphogenetic protein 4) in hESC line, CHA15 hESC results in KDR+ mesoderm specific differentiation. To determine whether the KDR+ population derived from hESCs enhances potential to differentiate along multipotential mesodermal lineages than undifferentiated hESCs, we analyzed the development of the mesodermal cell types in human embryonic stem cell differentiation cultures. In embryoid body (EB) differentiation culture conditions, we identified an increased expression of KDR+ population from BMP4-stimulated hESC-derived EBs. After induction with additional growth factors, the KDR+ population sorted from hESCs-derived EBs displays mesenchymal, endothelial and vascular smooth muscle potential in matrix-coated monolayer culture systems. The populations plated in monolayer cultures expressed increased levels of related markers and exhibit a stable/homologous phenotype in culture terms. In conclusion, we demonstrate that the KDR+ population is stably isolated from CHA15 hESC-derived EBs using BMP4 and growth factors, and sorted KDR+ population can be utilized to generate multipotential mesodermal progenitors in vitro, which can be further differentiated into cardiovascular specific cells.

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.