막걸리의 synbiotics 기능성을 향상시키기 위하여 유산균을 선발하고 이를 이용한 막걸리 최적 발효공정을 개발하였다. 막걸리에서 분리한 유산균은 Lactobacillus casei,Lactobacillus paracasei, Lactobacillus plantarum, Lactoba-cillus brevis와 Pediococcus acidilactici의 5종의 균주로 확인되었으며, 이들 중 발효특성, pH 내성 및 담즙산염 내성평가를 통해 막걸리의 probiotics로서 Lactobacillus planta-rum JS1 (KFCC11566P)를 최종 선발하였다. 또한 최적 발효조건 설정을 위해 누룩과 입국의 발효제 사용여부, 원료의 증자여부, 발효조건 등을 토대로 발효과정 중 효모의 생육을 저해하지 않으면서 이상발효 현상을 보이지 않고 최종 L. plantarum JS1의 균체량이 높은 공정을 설정하였다.최종 개발된 공정은 L. plantarum JS1을 쌀의 0.012% 첨가량으로 효모와 동시에 접종하고, 발효제로 입국을 사용하며, 무증자쌀을 이용, 25oC에서 발효시키는 방법으로, 이를 막걸리의 synbiotics 기능향상을 위한 최적 공정으로 설정하였다.

MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading or repressing targeted mRNAs. These molecules are about 21-25 nucleotides in length and exert their effects by binding to partially complementary sites in mRNAs, predominantly in the 3'-untranslated region (3'-UTR). Recent evidence has demonstrated that miRNAs can function as oncogenes or tumor suppressors through the modulation of multiple oncogenic cellular processes in cancer development, including initiation, cell proliferation, apoptosis, invasion and metastasis. In our present study, we examined the expression profile of miRNAs related to oral cancer cell growth inhibition using normal human oral keratinocytes (NHOK) and YD-38 human oral cancer cells. By miRNA microassay analysis, 40 and 31 miRNAs among the 1,769 examined were found to be up- and down-regulated in YD-38 cells compared with NHOK cells, respectively. Using qRT-PCR analysis, the expression levels of miR-30a and miR-1246 were found to be increased in YD-38 cells compared with NHOK cells, whereas miR-203 and miR-125a were observed to be decreased. Importantly, the overexpression of miR-203 and miR-125a significantly inhibited the growth of YD-38 cells. This finding and the microarray data indicate the involvement of specific miRNAs in the development and progression of oral cancer.

Recently, mini-implant is popular in the orthodontic treatment due to its simplicity and convenient surgical procedure. The objective of this study is to provide the anatomical guideline for mini-implant placement by analysing the cortical bone thickness in Korean. Hemi-sections of sixteen maxillae and twenty-two mandibles with normal teeth were used. Interdental areas between the 1st premolar and the 2nd premolar (Group 1), the 2nd premolar and the 1st molar (Gruop 2), and the 1st molar and the 2nd molar (Group 3) were sectioned and then scanned. After setting the axis of teeth, the cortical bone thickness was measured at the distance of 2 mm, 4mm, 6 mm, and 8 mm from alveolar crest. The mean thickness of cortical bone in the maxilla according to distance from alveolar crest was 1.30 ± 0.63 mm (2 mm), 1.49 ± 0.62 mm (4mm), 1.72 ± 0.64 mm (6mm), and 1.90 ± 0.90 mm (8 mm) at the buccal side and 1.33 ± 0.47 mm, 1.31 ± 0.45 mm, 1.37 ± 0.55 mm, and 1.39 ± 0.58 mm at the palatal side. In the mandible, that was 3.14 ± 1.71 mm, 4.31 ± 2.22 mm, 4.23 ± 1.94 mm, and 4.30 ± 1.57 mm at the buccal side and 1.98 ± 0.88 mm, 2.79 ± 1.01 mm, 3.35 ± 1.27 mm, and 3.93 ± 1.38 mm at the lingual side. The buccal cortical bone thickness in the maxilla was decreased from Group 1 to Group 3, while the thickness of palatal side was no change. In the mandible, it did not show a tendency at the buccal side and it was decreased from Group 1 to Group 3 without significant difference at the lingual side. Therefore, the buccal side of the Group 1 and Group 2 in both the maxilla and mandible seems to be the most appropriate site for a mini-implant placement with taking the stability and retention.

Angelica decursiva has been used in Korean traditional medicine as an antitussive, an analgesic, an antipyretic and a cough remedy. However, its anti-cancer properties have not yet been well defined. In our current study, we report the cytotoxic activity and the mechanism of cell death induced by ethanol extracts of Angelica decursiva (EEAD) against the human oral cancer cell line, KB. Treatment of KB cells with EEAD induced apoptotic cell death in both a dose- and time-dependent manner as determined by MTT assay and DNA fragmentation. However, no cytotoxic effects of EEAD against human normal oral keratinocytes (HNOK) were evident. By western blot analysis, we found that apoptosis in KB cells is associated with a decrease in procaspase-7 and -9. In addition, the activation of caspase-7 was detectable in living KB cells by fluorescence microscopy. These results suggest that EEAD exhibits anti-cancer activity in KB cells via apoptosis and thus has potential as an anticancer agent in future drug development strategies.

Angelica decursiva has been used in Korean traditional medicine as an antitussive, an analgesic, an antipyretic and a cough remedy. However, the anti-cancer properties of Angelica decursiva have not yet been well defined. In our current study the cytotoxic activity of ethanol extracts of Angelica decursiva root (EEAD) and the mechanism of cell death exhibited by EEAD were examined in FaDu human head and neck squamous cell carcinoma cells. The cytotoxic effects of EEAD upon the growth of FaDu cells were examined with an MTT assay. In addition, the mechanism of cell death induced by EEAD was evaluated by DNA fragmentation analysis, immunoblotting and caspase activation measurements. EEAD induced apoptotic cell death in FaDu cells in a concentration- and time-dependent manner, as determined by MTT assay and DNA fragmentation analysis. Furthermore, the proteolytic processing of caspase-3, -7 and -9 was increased by EEAD treatment of FaDu cells. In addition, the activation of caspase-3 and -7 was detected in living FaDu cells by fluorescence microscopy. These results suggest that EEAD can induce apoptosis and suppress cell growth in cancer cells and may have utility as a future anticancer therapy.

Early growth response 1 (Egr1) is an immediate early response gene which is induced by various external stimuli and acts as transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. It is well known that Egr1 regulates transcription of a cluster of genes in cancers and luteinizing hormone (LH) beta subunit in the pituitary. In addition to function of Egr1 in cancers and pituitary, we recently showed that Egr1 acts as a local master regulator to mediate estrogenic actions in the uterus. However, regulatory mechanism by which Egr1 directs transcription of its downstream target genes in the uterus remains to be yet explored. Thus, we have tried to identify direct target genes of Egr1 in the uterus by analyzing mRNA microarray data sets followed by in silico promoter analyses with chromatin immunoprecipitation (CHIP). mRNA expression profiles of Egr1(－/－) uteri and Egr1(－/－) ovaries were compared to those of wildtype mice to provide a potential list of direct target genes of Egr1 in the uterus. Whereas Egr1 is rapidly and transiently induced in the ovary and the uterus by external stimuli, LH and estrogen, respectively with a similar manner, a list of differentially expressed genes between Egr1(+/+) and Egr1(－/－) mice were barely overlapped between these two datasets. This result suggests that the transcriptional network of Egr1 in the uterus is quite different from that in the ovary. The list of differentially expressed genes in Egr1(－/－) uterus was enriched by RT-PCR. In silico analyses with MatInspector provided evidence that Egr1 binding sites are relatively enriched in －500 bp promoter regions of genes in the list. CHIP assays for Egr1 antibody with uterine tissues 2 h after estrogen treatment reinforced the possibility that genes identified in this study such as Gadd45g and Lbh could be directly regulated by Egr1 in uterine context. Collectively, we show that bioinformatic analyses of expression profiles with in silico analyses could be a useful tool to enrich potential candidates of direct target genes of transcription factors.

Early growth response 1 (Egr1) belongs to the Egr family of zinc finger transcription factors that regulates cell growth, differentiation, and apoptosis. Egr1(－/－) female mice are infertile due to anovulation resulting from luteinizing hormone β subunit (LHβ) deficiency. While it is clear that Egr1 is a critical factor to regulate transcription of LHβ in the pituitary gland, function of Egr1 and mechanisms by which estrogen (E2) and/or progesterone (P4) regulates Egr1 in uterus still remain unexplored. Using multiple approaches, here we have characterized regulatory mechanism of Egr1 induction in the uterus and uterine phenotypes of Egr1(－/－) mice. Eight-week-old female mice were ovariectomized (OVX) and rested for a week. Uteri of OVX mice treated with various concentrations of E2 and/or other hormones were collected at 2h after hormone treatment unless otherwise indicated. Collected uteri were utilized for mRNA microarrays, realtime-RT-PCR, Western blotting, and histological analyses for immunofluorescence and BrdU staining. Egr1 mRNA was rapidly induced with the highest level at 2h after E2 treatment and gradually decreased to basal levels at 12 h. E2-induced phosphorylation of ERK1/2 and AKT, and Egr1 transcription were effectively inhibited by pretreatment of ICI 182,780. Pharmacological inhibition of ERK1/2, but not AKT significantly blocked E2-induced Egr1 expression in the uterus. P4 effectively dampened E2-dependent Egr1 transcription and its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, BrdU incorporation experiments provided evidence that epithelial cells undergo hyperproliferation in Egr1(－/－) mice. This is consistent with microarray data that several key factors for cell cycle progression such as cyclin Ds and E2F1 are overexpressed in these mice. Furthermore, in the uteri of OVX Egr1(－/－) mice treated with E2+P4, stromal cell proliferation is severely impaired and epithelial cells persistently proliferating. While ovulation, fertilization and embryo development normally occur in Egr1(－/－) mice treated with a superovulation regime to rescue LH deficiency, embryo implantation is severely impaired. Blastocysts were not able to implant even on day 6 of pregnancy in Egr1(－/－) mice. In addition to embryo implantation, uterine response to artificial decidualization in hormone-primed Egr1(－/－) OVX mice was relatively less than that of wildtype mice. Collectively, our results show that Egr1, which is rapidly induced by E2-ER-ERK1/2 pathways, is a critical factor to control E2-dependent cell proliferation via regulation of a spectrum of genes for embryo implantation in the uterus.

MicroRNAs (miRNAs) are single-stranded, small non-coding RNAs which are critical for gene regulatory networks by directing the translational repression or degradation of complementary target mRNAs. They can be divided into two subclasses: canonical and non-canonical miRNAs. Canonical miRNAs are produced from long primary transcripts by sequential complex events in which RNA III enzymes such as Drosha and Dicer and accessory factors such as DGCR8 and Argonautes work cooperatively. DGCR8 is a RNA-binding protein that assists Drosha to process canonical miRNAs in the nucleus. To understand function of canonical miRNAs in uterine physiology, we have characterized uterine phenotypes of uterine-specific DGCR8 knock-out mice (uDGCR8 KO, DGCR8flox/flox; PRcre/+), and hormonal regulation of expression profiles of major factors working for miRNA biogenesis in the uterus. Gross morphological and histological analyses, immunohistochemistry, PCR and realtime RT-PCR were performed. While DGCR8 and Drosha do not seem to be regulated by ovarian steroid hormones, expression of Dicer, Exportin 5 and Argonaute 2 was transiently increased by E2 but not by P4. Combination of E2+P4 did not have any additional effects on their expression profiles. Genomic DNA PCR analyses showed that while DGCR8 gene is not completely deleted in the uterus, DGCR8 is specifically deleted in the uterus where PR is strongly expressed. uDGCR8 KO females bred with fertile males did not produce any offspring, suggesting that these mice are infertile. Vaginal smear analyses provided evidence that these mice do not undergo estrous cycle. Whereas gross morphology and histological analyses of uteri of 3-week-old uDGCR8 KO mice is similar to that of DGCR8flox/flox mice (control), uteri of 5- and 8-week-old mice are extremely thinner and shorter than those of control mice. These results were supported by significant decrease in uterine weight/body weight of uDGCR8 KO mice at 5-week-old age onward. Interestingly, this phenotype is reflected by significant increase of PR expression in the uteri of 4-week-old mice. Expression of DGCR8 and Dicer is significantly increased after birth. BrdU incorporation experiments showed that cell proliferation governed by ovarian steroid hormones does not normally occur in these mutant mice. Furthermore, artificial decidualization does not occur in these mice. Collectively, these results conclude that canonical miRNAs plays critical roles in normal uterine development and steroid hormone-dependent uterine function.