Ganoderma lucidum has been traditionally used as a medicine for treatment of bronchitis, arthritis, and high blood pressure, and it has been reported to display many biological activities including anticancer and immune activities. Since mushroom mycelium is known to have excellent biological activities together with mushroom fruiting body, studies on biological activities of mushroom mycelium have been actively conducted. Thus, the present study compared the biological activities before and after the cultivation of Ganoderma lucidum mycelium on Atractylodes rhizoma. When the radical scavenging activity was assessed by the DPPH assay, ARGL (ethanol extract of Atractylodes rhizoma mycelium fermented with Ganoderma lucidum) showed radical scavenging activity of 5.58~82.56% at concentrations of 10~500 μg/assay, while AR (ethanol extract of Atractylodes rhizoma) showed radical scavenging activity of 5.27~72.08% at the same concentrations. When measured by using the ABTS assay, ARGL showed higher radical scavenging activity than AR, which was consistent with the result obtained by the DPPH assay. In the MTT assay, the cytotoxicity of ARGL against all cell lines was higher than that of AR. In particular, the cytotoxicities of AR and ARGL against Hep3B at a concentration of 400 μg/assay were 71.81% and 86.40%, respectively. In addition, the result obtained by the SRB assay was consistent with the result obtained by the MTT assay. According to the results mentioned above, there is a high probability that medicinal herb cultures using mycelium can be used as sources of functional foods since the cytotoxicities against cancer cells and antioxidant activities increased when the mycelium was fermented with Atractylodes rhizoma.

본 연구는 갈근 추출물의 항산화 활성 및 멜라닌세포 효과를 평가하기 위하여 총 폴리페놀 함량, 총 플라보노이드 함량, DPPH radical 소거 활성을 통하여 항산화 활성을 살펴보고, B16F10 melanoma 세포에 대한 세포 독성 및 멜라닌 생합성 억제능 효과를 측정하였다. 연구 결과 B16F10 melanoma 세포에 대해 독성을 나타내지 않았으며, B16F10 melanoma 세포에 α-MSH로 멜라닌 생성 을 유도한 후 멜라닌 생합성 억제능을 측정한 결과 멜라닌의 생성 증가가 농도 의존적으로 억제되는 것 을 확인하였다. 항산화 활성에 대한 결과로는, 갈근 추출물은 폴리페놀과 플라보노이드의 함량이 높아 추출물의 농도가 증가함에 따라 DPPH radical 소거 활성이 증가되는 것을 확인하였다. 이와 같은 결과 를 통하여 갈근 추출물이 항산화 활성과 멜라닌세포 대한 멜라닌생성억제 효과가 뛰어나고 피부 세포에 대한 독성이 낮으며, 피부의 멜라닌 세포에 대한 안전성이 확인됨에 따라 화장품 소재로서의 가능성을 확인하였다.

Although Semisulcospira libertina is generally regarded as a supplement for the alleviation of alcohol hangover, little is known about its effects on cell metabolism. Therefore, this study was conducted to analyze the constituents of the extracts prepared using different extraction methods and to compare their biochemical properties. The amino acid contents were found to be much higher in acidic and enzymatic hydrolysates than hot water extracts from S. libertina. DPPH radical scavenging activities in acidic and enzymatic hydrolysates were higher than those of hot water extracts. Three types of S. libertina hydrolysate was added to HepG2 cells damaged by acetaminophen (AAP), after which the survival rate of HepG2 cell were measured. In addition, lactate dehydrogenase (LDH) activities in the culture media were evaluated. The survival rates of HepG2 cells were 77.0±4.3% and 81.5±1.3% at 3 h and 5h enzymatic hydrolysates, respectively. These cell survival rates were higher compared to those of the negative control group (67.8±4.3%) treated only with acetaminophen. Cellular toxicities induced by treatment with AAP were also significantly alleviated in response to treatment with the extracts of S. libertina. In addition, the activities of 2 key enzymes that metabolize ethanol, alcohol dehydrogenase and aldehyde dehydrogenase, were upregulated by 4.7- and 2.7-fold respectively in response to treatment with a 3 h enzymatic hydrolysate of S. libertina. Taken together, these results provide biochemical evidence of the method by which S. libertina exerts its biological functions, including the alleviation of alcohol hangover and the protection of liver cells against toxic insults.

Although Semisulcospira libertina is generally regarded as a supplement for the alleviation of alcohol hangover, little is known about its effects on cell metabolism. Therefore, this study was conducted to analyze the constituents of the extracts prepared using different extraction methods and to compare their biochemical properties. The amino acid contents were found to be much higher in acidic and enzymatic hydrolysates than hot water extracts from S. libertina. DPPH radical scavenging activities in acidic and enzymatic hydrolysates were higher than those of hot water extracts. Three types of S. libertina hydrolysate was added to HepG2 cells damaged by acetaminophen (AAP), after which the survival rate of HepG2 cell were measured. In addition, lactate dehydrogenase (LDH) activities in the culture media were evaluated. The survival rates of HepG2 cells were 77.0±4.3% and 81.5±1.3% at 3 h and 5h enzymatic hydrolysates, respectively. These cell survival rates were higher compared to those of the negative control group (67.8±4.3%) treated only with acetaminophen. Cellular toxicities induced by treatment with AAP were also significantly alleviated in response to treatment with the extracts of S. libertina. In addition, the activities of 2 key enzymes that metabolize ethanol, alcohol dehydrogenase and aldehyde dehydrogenase, were upregulated by 4.7- and 2.7-fold respectively in response to treatment with a 3 h enzymatic hydrolysate of S. libertina. Taken together, these results provide biochemical evidence of the method by which S. libertina exerts its biological functions, including the alleviation of alcohol hangover and the protection of liver cells against toxic insults.

Coffee (Coffea spp.) is one of the most important agricultural commodities, being widely consumed in the world. Various beneficial health effects of coffee have been extensively investigated, but data on habitual coffee consumption and its bio-physiological effect have not been clearly explained as well as it is not proved the cause and effect between drinking coffee and its bio-physiological reactions. We made the dialyzed coffee extract (DCE), which is absorbable through gastrointestinal tract, in order to elucidate the cellular effect of whole small coffee molecules. RAW 264.7 cells, a murine macrophage lineage, were directly treated with DCE, i.e., DCE-2.5 (equivalent to 2.5 cups of coffee a day), DCE-5, and DCE-10, for 12 hours, and their protein extracts were examined by immunoprecipitation high performance liquid chromatography (IP-HPLC). RAW 264.7 cells differently expressed the inflammation-related proteins depending on the doses of DCE. RAW 264.7 cells treated with DCE showed marked increase of cathepsin C, cathepsin G, CD20, CD28, CD31, CD68, indicating the activation of innate immunity. Particularly, the macrophage biomarkers, cathepsin G, cathepsin C, CD31, and CD68 were markedly increased after DCE-5 and DCE-10 treatments, and the lymphocyte biomarkers, CD20 and CD28 were consistently increased and became marked after DCE-10 treatment. On the other hand, RAW 264.7 cells treated with DCE showed consistent increase of IL-10, an anti-inflammatory factor, but gradual decreases of different pro-inflammatory proteins including TNFα, COX-2, lysozyme, MMP-2, and MMP-3. In particular, the cellular signaling of inflammation was gradually mitigated by the reduction of TNFα, COX-2, IL-12, and M-CSF, and also the matrix inflammatory reaction was reduced by marked deceases of MMP-2, MMP-3, and lysozyme. These anti-inflammatory expressions were consistently found until DCE-10 treatment. Therefore, it is presumed that DCE may have dynamic effects of innate immunity activation and pro-inflammation suppression on RAW264.7 cells simultaneously. These effects were consistently found in the highest dose of coffee, DCE-10 (equivalent to 10 cups of coffee a day in man), that might imply the small coffee molecules were accumulated in RAW 264.7 cells after DCE-10 treatment and produce synergistic cytokine effects for innate immunity activation and anti-inflammatory reaction concurrently.

Although salivary gland adenocarcinoma NOS accounts for third prevalence rate of all salivary gland tumors, it is one of the most aggressive solid tumors. Current therapy does not significantly improve survival rates. Thus, investigating new therapeutic modalities against salivary gland adenocarcinoma NOS is necessary. It is well known that docetaxel(TXT) as an antimicrotubulin agent induces mitotic block in proliferating cells. TXT has significant antitumor effects, and it is currently being tested in patients with malignant tumors, but TXT has not yet been tested in malignant salivary gland tumors. The purpose of this study were to examine the effects of TXT and to evaluate the biological mechanisms of TXT on salivary gland adenocarcinoma NOS. Proliferation, cell cycle regulation, connexin43 expression, apoptosis, and Fas receptor(FasR) expression were measured in cultured SGT cell line. Proliferation was little changed after 10ng/ml TXT exposure, but cellular proliferation was inhibited according to increasing concentration of TXT and time. Especially it was prominently inhibited after 96 hrs at 20ng/ml. G2-M arrest stage showed about up to 5 fold increase after exposure of TXT by flow cytometry. Apoptosis index showed about up to 8 fold increase after exposure of TXT by flow cytometry. Fas expression showed about up to 3 fold increase after exposure of TXT by flow cytometry. Apoptosis showed about up to 3 fold increase at 20ng/ml after exposure of TXT and anti-Fas agonist by flow cytometry. In Immunoslot blotting, Cx 43 protein expression was increased after TXT treatment. It suggested that TXT might induce apoptosis in SGT cells and could be used as a potent and specific chemotherapeutic tool for the treatment of salivary gland adenocarcinoma NOS in future.

To gain insights into the role of purinergic receptors in human dental pulp cells (hDPCs) differentiation, we characterized the expression and functional activity of P2Y1 receptors and investigated the effects of ADP on the proliferation and differentiation of this pulp stem-like cell population. Our data showed that ADP did not induce cell proliferation to expose the various ADP concentrations for 72 hours, but the proliferative capacity of hDPCs was inhibited at higher ATP concentrations (100 μM). Using RT-PCR analysis, we found that ADP induced several P2Y receptors including P2Y1 as well as odontoblastic differentiation genes, dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) in a dose-dependent manner. The effects of ADP on the expression of DMP-1 and DSPP mRNA were prevented by the P2Y1 antagonist MRS2179. The extracellular matrix calcium deposits were clearly observed in ADP-treated hDPCs by alizarin red S staining. Quantitative measurement of mineralization induced by ADP was significantly inhibited in MRS2179-treated hDPCs. These results may provide new insights into the molecular regulation of the differentiation of hDPCs.

목적: 미세먼지 등의 증가로 안구세척제 사용이 늘어감에 따라 안구세척제의 자극성에 대한 연구가 필요하므로 안구세척제의 세포독성을 평가하였다. 방법: 세포독성 검정을 위해 L929 세포는 α-MEM (GIBCOCo, USA)에 10% FBS, fungizone 및 antibiotics를 첨가해 배양하여 96 well-plate에 각각 분주하고 세포가 well 당 80% 정도 채워졌을 때 처리하였다. 처리된 세포는 MTT 200 ㎍/㎖가 포함된 배양액으로 교환하고 3시간 반응시켰다. 3시간 후 배양액을 버리고 dimetylsulfoxide(DMSO; Sigma CO. USA)를 200㎕/well 씩을 넣어 5분간 실온 방치하여 푸른색 결정인 formazan을 용해 시킨 후 ELISA plate reader (Bio-Tech, USA)로 570nm에서 흡광도를 측정하여 대조군과 비교하여 세포 증식 저해율이 30% 이상이면 독성이 있는 것으로 판정하였다. 결과: 본 연구의 실험결과는 저자극 안구세척제에 대한 시험성적은 세포생존율 50%를 기준으로 높을수록 저자극, 낮을수록 자극성을 보이는데 시중에 가장 많이 유통되고 있는 안구세척제의 세포독성실험을 한 결과, 65.48±2.25%로 나타나 자극이 아예 없다고는 볼 수 없으나 자극은 낮은 것으로 나타났다. 결론: 본 연구는 환경오염으로 인한 안구세척제의 개발의 중요성을 알고 눈의 건강을 위해 더 지속적으로 안구에 저자극인 안구세척제를 개발이 있어야 할 것으로 사료된다.

돼지 난자의 체외배양 과정에서 배양액의 삼투압의 차이 및 glycine과 alanine 단독 또는 병행 첨 가가 단위발생 및 체세포 핵이식 난자의 배 발육에 미치는 영향을 검토하였다. 난자 의 체외성숙 에는 10% 돼지 난포액, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin이 첨가된 TCM-199을 이용하였다. 체외성숙 42∼44시간 후 제1극체가 방출된 난자만을 선별하여 단위발생 및 체세포 핵이식 난자를 생산한 후 modified porcine zygote medium (mPZM)-3 배양액에서 7일간 배양하였다. 실험 설계에 따라 체외배양 7일 또는 체외배양 초기 48시간 동안 280 또는 320 mOsm의 삼투압에서 난자를 배양하였다. 실험1에서 체외배양 7일 동안 280 또는 320 mOsm의 배 양액 내 glycine 또는 alanine 첨가 효과를 검토한 결과 삼투압 차이에 따른 분할률 및 배반포 발달 률에는 차이를 보이지 않았으나 320 mOsm에서 배양된 난자 및 280 mOsm에 glycine을 첨가한 군 은 280 mOsm에서 배양된 난자에 비해 높은 배반포 세포수를 보였다 (36.5-38.0 vs. 31.1, P<0.05). 실험2에서는 체외배양 초기 48시간 동안 280 또는 320 mOsm의 삼투압에서 배양하였고, 그 후 280 mOsm 배양액으로 옮겨 5일간 추가로 배양하였다. 또한 체외배양액 내 4.1 mM glycine 첨가가 배 발육에 미치는 효과를 검토하였다. 단위발생 난자를 배양초기 48시간 동안 320 mOsm에서 glycine이 첨가된 배양액에서 배양한 군이 280 mOsm에서 배양한 군에 비해 유의적으로 높은 배반 포 발달율을 보였다(36.5-50.4% vs. 25.9-27.9%, P<0.05). 또한 배양 초기 48시간 동안 320 mOsm 배 양액에 glycine을 첨가하여 배양한 난자 (50.4%)가 다른 처리군의 난자(25.9-36.5%)에 비해 유의적 으로 높은 배반포 발달율을 보였다(P<0.05). 실험3에서는 실험2와 동일한 실험설계로 체세포 핵이 식으로 작성된 배반 포의 inner cell mass (ICM)와 trophectoderm (TE) 세포수를 조사하였다. 실험결과 초기 46시간 동안 glycine이 첨가된 320 mOsm 처리군에서 배양된 난자의 ICM 비율이(26.0%) 280 mOsm 삼투압에 glycine을 첨가한 군(17.8%)에 비해 유의적(P<0.05)으로 증가하였다. 본 연구결과 단위 생식 및 체세포 핵이식 난자의 체외배양에서 glycine의 첨가 및 체외 배양초기 48시간 동안 320 mOsm 삼투압 처리는 배 발육과 배반포 세포수를 증가시키는 것으로 확인되었다.

Aloe-emodin (AE) is the major bioactive component in aloe and known to exhibit anti-inflammatory activities. However, it has not been elucidated whether its anti-inflammatory potency can contribute to the elimination of obesity. The aim of the current study is to investigate the effect of AE on toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with AE (0-20 μM) for one hour, followed by LPS treatment for 30 min and then, adipokine mRNA expression levels were measured. Next, TLR4-related molecules were measured in LPS-stimulated 3T3-L1 adipocytes. AE significantly decreased the mRNA expression of the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) in a dose-dependent manner. Moreover, AE suppressed TLR4 mRNA expression. Further study showed that AE could suppress the nuclear factor-κB (NF-κB) and phosphorylation of extracellular receptor-activated kinase (pERK). The results of this study suggest that AE directly inhibits TLR4/NF-κB/ERK signaling pathways and decreases the inflammatory response in adipocytes.

7년 이상 장기간 배양으로 인한 고가의 산양삼은 항염증에 효과가 있다고 잘 알려져 있다. 하지만, 재배기간에 따른 항염증능의 비교 연구는 부족한 실정이다. 본 연구는, 3년, 5년, 7년 근령의 산양삼 추출물을 이용하여 RAW264.7 대식세포의 염증반응 모델에서의 항염증능을 비교분석하였다. 이를 위하여, 염증성 싸이토카인 (IL-6, TNF-α)의 분비, 표면 단백질(CD80, CD86, MHC-Ⅱ)의 발현 및 대식능을 분석하였다. 각 근령의 산양삼 추출물을 0.1, 1, 10ppm 농도로 RAW26.47 세포주에 처리한 후, LPS로 염증반응을 유도하였을 때, IL-6와TNF-α의 분비량이 모든 근령군에서 농도의 존적으로 감소함을 보였다. 하지만, 일반적으로 근령이 클수록 싸이토카인 분비 억제 효과가 높은 것으로 확인 되었다. 대식세포의 활성화마커인 표면 단백질의 발현양도 모든 실험군에서 농도의존적으로 감소 하는 경향을 나타내었다. 구체적으로는, CD80과 CD86의 발현은 3년삼, 5년삼, 7년삼의 10ppm에서 무처리 대조군과 비교하여 유의적으로 감소하였고, 근령간의 차이는 보이지 않았다. MHC-Ⅱ의 발현은 일정한 경향은 나타나지 않았고 3, 7년삼의 10ppm처리군에서 감소하였다. 대식능은 3년삼의 10ppm을 제외하고는 유의적 차이를 보이지 않았다. 이러한 결과는 산양삼 근령에 증가에 따라 항염증능이 증가하지만, 대식능에는 영향을 미치지 않는 것으로 평가되며 동물실험을 통하여 확증할 예정이다.

옥수수수염은 다양한 플라보노이드를 함유하고 있어 항산화능이 보고되었으며, 전통의학에서는 부종의 제어에 효과가 있는 것으로 알려져 있다. 본 연구는 LPS로 자극된 RAW 264.7 대식세포의 염증모델에서, 옥수수수염 추출물의 항염증기작을 탐색하였다. 옥수수수염 추출물과 함께 배양한 RAW 264.7 대식세포의 대식능, 항원제시 단백질 (CD80, CD86, MHC class II)의 발현량, 염증성 유전자 (iNOS, COX-2, TNF-α, IL-6)의 전사 및 염증성 싸이토카인 (TNF-α, IL-6) 분비능을 측정하였다. FITC-latex beads를 이용하여 대식능을 측정한 결과, 옥수수수염 추출물을 처리함에 따라 유의적인 차이를 보이며 농도 의존적으로 증가하는 경향을 나타내었다. 또한, 형광항체를 이용하여 LPS 자극에 따른 항원제시 단백질의 발현량을 flow-cytometer로 측정하였다. 그 결과, CD80 단백질은 옥수수수염 추출물을 처리하였을 시 농도에 관계 없이 그 양이 유의적으로 증가하였으며, CD86 단백질은 농도 의존적으로 유의적인 차이를 보이며 증가하였다. 대식세포의 대표적인 염증성 신호전달체계인 NF-kB의 down-stream 유전자의 전사량을 qRT-PCR을 이용하여 측정한 결과, iNOS, COX-2, TNF-α 및 IL-6의 전사량은 모두 유의적으로 감소하였다. 염증성 싸이토카인인 TNF-α와 IL-6의 전사량의 변화가 세포배양액 내 분비량과의 상관관계가 있는지 확인하기 위하여 ELISA법을 이용하여 배양액 내 싸이토카인을 정량한 결과, 모두 유의적으로 감소함을 보였다. 따라서, 본 연구결과는 옥수수수염 추출물의 항염증 기능을 밝히고 있다. 더불어, 대식세포의 후천성면역을 매개하는 항원제시능은 영향을 받지 않음으로서 면역저하효과는 없을 것으로 판단된다.

Coffee is one of the most familiar beverages to modern human adults, but its bio-physiological effect has not been clearly elucidated. It was known that more than one thousand chemicals were included in the ordinary coffee extract. Among them, the caffein and chlorogenic acid (caffeoylquinic acids) are most abundant and have been investigated by many authors so far. In order to know the real cellular effect of whole coffee extract elements, the dialyzed coffee extract (DCE)1) was made to get coffee elements less than 1000 Da molecular weight, which are freely absorable through gastrointestinal tract. It was directly treated in the culture of RAW 264.7 cells, a murine macrophage lineage. RAW 264.7 cells were treated with DCE equivalent to 2.5 cups of coffee (DCE-2.5), DCE-5, and DCE-10 for 12 hours, and their protein extracts were examined by histological observation and immunoprecipitation high performance liquid chromatography (IP-HPLC). RAW 264.7 cells differently expressed the proliferation-related proteins depending on the dose of DCE. DCE-2.5 and DCE-5 enhanced the cellular growth of RAW 264.7 cells by increasing the expression of β-actin, PCNA, Ki-67, MPM2, MAX, cMyc, E2F-1, and Rb-1, and by decreasing the expression of MAD and p21. These proliferation-related proteins were rarely affected by DCE-10. DCE-2.5 and DCE-5 induced the cellular proliferation of RAW 264.7 cells by the signaling of E2F-1 and cMyc, respectively, but these cellular effects almost disappeared in DCE-10. Therefore, it was presumed that the low dose of coffee, DCE-2.5 and DCE-5 might be effective for the proliferation of murine macrophages, RAW264.7 cells, contrast to the high dose of coffee, DCE-10. It was also suggested that the low dose of DCE-2.5 and DCE-5 be helpful to increase the innate immunity in vivo by increasing the cell number of macrophages in contrast to the high dose of DCE-10.

복섬, Takifugu niphobles 부화 자어를 대상으로 기아에 따른 성장, 생존 및 간세포 핵 크기에 미치는 영향을 조사하였다. 자어의 전장은 부화 후 3일 까지 증가하였으나 부화 후 3일에서 부 화 후 6일까지 절식구는 공급구에 비해 유의한 낮은 성장을 보였다. 공급구 복섬의 체중은 실 험 기간 중 지속적인 증가를 보였다. 절식구의 체중은 부화 후 3일 까지 증가하였으나, 부화 후 3일 이후는 서서히 감소하였다. 절식구의 생존율은 공급구의 생존율에 비해 유의하게 낮았 으며(p < 0.05) 부화 후 6일째 절식구 자어는 폐사하였다. 절식구의 간세포 핵 크기는 부화 후 3일 까지는 공급구의 간세포 핵 크기와 유의한 차이가 없었으나, 부화 후 4일에서 부화 후 6일 까지는 절식구은 공급구에 비해 간세포 핵 크기 감소를 보이며 조직상 차이를 보였다. 본 연구 결과 도출된 자료들은 복섬의 영양상태 지표로 사용될 수 있을 것으로 사료된다.

본 연구는 우엉 뿌리 추출물의 항산화 활성 및 피부에 대한 안전성을 평가하기 위하여 총 폴 리페놀 함량, 총 플라보노이드 함량, DPPH radical 소거 활성을 통하여 항산화 활성을 살펴보고, B16F10 melanoma 세포에 대한 세포 독성 및 자외선 A에 대한 피부 세포 보호 효과를 확인하였다. 또 한 화장품 소재로서의 활용을 검증하기 위하여 1차 피부 첩포 테스트를 실시하였다. 본 실험 결과 우엉 뿌리 추출물의 함량이 증가됨에 따라 높은 폴리페놀과 플라보노이드의 함량이 확인되었으며, DPPH radical 소거 활성을 확인하였다. B16F10세포에 대한 세포 독성을 확인한 결과 B16F10 melanoma 세포 에 대해 독성이 낮고, 자외선 A에 대해 80% 이상의 세포 보호 효과를 확인하였다. 또한 1차 피부 첩포 테스트를 통해 우엉 뿌리 추출물이 피부에 자극이 거의 없음을 확인하였다. 이러한 결과를 통하여 우엉 뿌리 추출물이 항산화 활성과 자외선에 대한 피부 보호 효과가 뛰어나고 피부 세포에 대한 독성이 낮으 며, 피부에 대한 안전성이 확인됨에 따라 화장품 소재로서의 가능성을 확인하였다.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

Recently, the area of marine resources has become concerned with sources for the next generation of the bio-industry. Until present, development of the marine resources has remained limited, although a large number of these resources are considered to have potential for various significant biological activities. Most marine sponges, marine algae and coral could be used to create specific compounds for survival against a harsh environment. Therefore, it was necessary that these materials needed to be elucidated with biological activities, such as like anti-inflammatory, anti-viral or anti-cancer effects for their utilization in the bio-industry. In this study, we screened extracts of marine resources for their anti-cancer effect on human colorectal cancer cells. These resources were collected at Kosrae of Micronesia on April, 2013 and extracted with methanol. Cytotoxicity of marine resources was observed. Of a total of 20 specimens, three specimens dose-dependently demonstration inhibition of cell viability. Furthermore, cells treated with these specimens for 48h were induced p53, p21, Bax and caspase-3. The results suggest that they involved p53-mediated apoptosis. Two positive specimens (1304KO-327 and 1304KO-329) were verified as the identical materials, which are Hyrtios sp. Unfortunately 1304KO-207 was not yet classified and needed to identify in the further study. There results suggested that marine resources with positive potential in anticancer effect would be good candidates as useful bio-resources.

Ameloblastic fibrosarcoma (AFS) is an extremely rare malignant odontogenic tumor characterized with benign ameloblastic cells islands and malignant mesenchymal component. While two-thirds of AFS seem to arise de novo, but one-third develops from recurrent ameloblastic fibroma (AF) or ameloblastic fibro-odontomas (AFO). Pathological distinction of malignant transformation is essential for appropriate treatment. The patient was a 28 years old man. Since the primary tumor was excised, the mass recurred 2 years later. The recurrent tumor was diagnosed as AFS. Chief complaint was pain in the right mandible. Computer tomography finding revealed multilocular intrabony lesion with radiopaque substance in the primary lesion. In the recurrent lesion cortical bone destruction was found. Microscopically, both the primary and recurrent lesions showed benign ameloblastic follicles with myxoid or highly cellular mesenchymal proliferation. The histological difference between primary and recurrent lesions were that foci of dental hard tissue composed of enamel and dentin were found only in the primary lesion, whereas nuclear pleomorphism was aggrevated in the recurrent lesion. The histological criteria determining malignancy were discussed.

Porphyromonas gingivalis is a gram-negative bacteria of rod shape, and grown in an anerobic condition. It colonizes in subgingival crevice and is known as a major pathogen causing chronic periodontitis. It possesses an invasive property and replicative potential within various cell types, presumably playing an important role in modulating biological behaviors of oral cancer. However, the pathophysiology of P. gingivalis in the malignant transformation of oral cancer has not been fully understood. In this study, we aimed to investigate molecular changes of oral squamous cell carcinoma cells induced by repetitive P. gingivalis infection that clinically resembles chronic periodontitis.

Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, was reported to various physiological and pharmacological effects including anti-cancer activity. Nevertheless, there is no report of the anticancer effect of lycorine in oral cancer cells. The effects of lycorine on cell proliferation and apoptosis were examined through trypan blue exclusion assay, 4’-6-diamidino-2-phenylindole (DAPI) stain, Live/Dead assay, Western blot analysis and RT-PCR. Lycorine suppressed cell viability and induced apoptosis in MC3 and HSC-3 cell lines. Lycorine decreased survivin protein but did not affect its mRNA. It regulated survivin through accelerating protein degradation in a time-dependent manner although neither proteasome nor lysosome was not associated with lycorine-mediated protein degradation. Collectively, our results suggest that lycorine may be a potential therapeutic anti-cancer drug candidate for the treatment of human oral cancer.