형질 전환 동물 생산에는 조직 및 시기 특이적 발현 조절이 가능하다는 장점 때문에 유즙 내로 외부 유전자를 발현시키는 시스템이 널리 이용되고 있다. 유전자 발현 즉, 단백질 생산은 프로모터의 강도뿐만 아니라 mRNA의 안정성에 의해서도 조절된다. 특히, polyadenylation에 의한 poly A의 길이는 in vivo와 올 in vitro에서 mRNA 안정성 및 목적 유전자의 번역효율에 영향을 준다. 본 연구에서는 이러한 mRNA 안정성이 목적 유전자의 발현에 미치는 영향을 알아보기 위해 3'-UTR 염기 서열을 분석하였다. 이 3'-untranslated region(UTR) 내의 poly A signal을 기준으로 putative cytoplasmic polyadenylation element(CPE) 부위와 downstream elements(DSE: U-rich, G-rich, GU-rich)의 염기 서열을 분석하고, 각각의 element를 기준으로 15 종의 luciferase reporter vector를 제작하여, 생쥐 유선 세포주(HC11)와 돼지 유선 세포주(PMGC)에 각각 transfection시킨 후 48시간 동안 배양하고 luciferase 발현량을 분석하였다. PMGC의 경우, luciferase의 발현은 exon 9의 CPE 2,3 및 DSE 1을 포함한 #6 construct에서 유의적으로 높은 발현량을 보였으며, exon 9의 CPE 2, 3과 DSE를 모두 포함하고 있는 #11 construct에서도 유의적으로 높은 발현량을 보였다. 이러한 결과는 형질 전환 돼지 생산에 있어 #6 및 11 construct의 사용은 목적의 유전자를 효과적으로 발현시키는데 기여할 것으로 사료된다.

Taxol(paclitaxel) is used in chemotherapy against several cancer. Treatment of tumor cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. The purpose of this study was to investigate apoptosis by the inhibitory effect of paclitaxel on the motility properties of human salivary gland adenocarcinoma cell lines. Paclitaxel inhibited cell motility induced by soluble and immobilized attractant. It suggested that paclitaxel would be a potent inhibitor of salivary gland adenocarcinoma cell motility independent of its cytotoxic and apoptotic activity.

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

Nuclear factor I-C (NFI-C) null mice demonstrated aberrant odontoblast differentiation, abnormal dentin formation, and thus molar lacking roots. However, the mechanism by which the disruption of NFI-C gene affect the expression of other genes in dental pulp cells remains unknown. In this study, in order to understand this mechanism, the gene expression of pulp cells in NFI-C deficient mice were compared to those of wild-type mice by cDNA microarray analysis. According to the cDNA microarray profile comparison, the disruption of NFI-C gene increased the expression of TGF-β and TGF-β receptor, whereas it decreased the expression of Smad proteins. Interestingly, most of the FGF-related genes were down-regulated in pulp cells by NFI-C gene disruption. Among the cell cycle-related genes, the expression of p16 and p18 were increased by NFI-C disruption, but the expression of cy clin E1 and cy clin D1 were decreased by NFI-C disruption. These results indicate that the disturbance of NFI-C gene suppressed the proliferation of pulp cells and up-regulated the expression of TGF-β and its downstream signaling molecules during root formation, contributing to the formation of short root containing abnormal dentin.

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The L-type amino acid transporter 1 (LAT1), an isoform of system L amino acid transporter, is highly expressed in cancer cells to support their continuous growth and proliferation. 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid(BCH) is a model compound for study of amino acid transporter as a system L selective inhibitor. We have examined the effect and mechanism of BCH on cell growth suppression in FaDu human head and neck squamous cell carcinoma. The BCH inhibited the L-leucine transport in a concentration-dependent manner with a IC 50 value of 43.8±4.3μM. The majority of L-leucine uptake is, therefore, mediated by LAT1 in FaDu cells. The growth of FaDu cells was inhibited by BCH in the time- and concentration- dependent manners. The formation of DNA ladder was not observed with BCH treatment in the cells. Furthermore, the proteolytic processing of caspase-3 and caspase-7 in the cells was not detected by BCH treatment. These results suggest that the BCH inhibits the growth of FaDu human head and neck squamous cell carcinoma through the intracellular depletion of neutral amino acids for cell growth without apoptotic processing

Biological organisms require iron for optimal metabolism. Intracellular pathogens also must secure iron especially during infection of animal hosts expressing NRAMP(natural resistance-associated macrophage protein), a transporter protein sequestering metal ions from pathogens. This study shows that extracytoplasmic function sigma factor σE is required for Salmonella virulence in NRAMP1-expressing mice, and further shows that iron deprivation turns on σE expression of Salmonella. The virulence of σE -deficient Salmonella is completely attenuated in C3H/HeN mice while wild type Salmonella kills all mice. Addition of an iron-chelator DTPA(Diethylene triamine pentaacetic acid) to culture media induces σE expression of Salmonella, but iron supplementation abrogates this induction. These findings suggest that iron limitation in host macrophages can trigger σE -dependent virulence system of Salmonella that may include bacterial iron homeostasis.

In this study, the apoptotic effects of the actin disruption agent, latrunculin B(LB) have been investigated on p53 deficient chronic myeloid leukemia cell line K562. A dose-dependent decrease in K562 cell proliferation was observed after LB treatment with maximum decrease in cell proliferation being at 1.5μM where the percent inhibition was 66.53%. F-actin stained with TRITC-phalloidin was shown as a peripheral ring or appeared diffusely distributed throughout the cytoplasm in untreated cells, this actin ring was decreased following LB treatment, and even large focal actin aggregates were formed. Treatment of K562 with LB(1.5μM) generated ROS substantially. LB activated expression in a dose-dependent manner. Therefore it can be concluded that LB, depolymerising agent of actin, induces apoptosis by producing ROS and up-regulating NF-kB and COX-2 activation.

Low energy photon irradiation by light in the far red to near infrared spectral range(630~1000nm) using low energy lasers or light emitting diode arrays has been found to modulate various biological processes in cell culture and animal models. The purpose of this study was to examine the light emitting diode irradiation effect on activity of normal human osteoblast on titanium plate in vitro by various energy density, and to observe morphologic change of NHost on titanium plate and to analysis concentration of Ca++, IP and ALP. NHost were cultured in DMEM containing 10% FBS, and observed by inverted microscope for attatchment to the surface of titanium plate. Ca++, I.P., and alkaline phosphatase( ALP) concentration in medium was calculated during 4 weeks, which was treated with Wilcoxon rank, Anova test and linear regression. Morphologic changes showed LED produced in vitro increases of cell growth of 144~256% in NHost. During a culture period, Ca++ concentration was decreased. LED treatment(>3J/cm2) stimulate calcium consumption in NHost. Statistically, a significant difference was not found between LED power density. LED treated group(>3J/cm2) had higher total inorganic phosphate concentrations than control group in NHost. Statistically, a significant difference was not found between LED power density. No significant changes were observed between ALP acitivity and LED treatment. In spite of LED power density, there were rapid growth rate of NHost and no significant of Ca++, IP and P concentration but these concentration showed predominant change than that of control.

The prominent side effect of cyclosporin A, immunosuppressive agent, in oral tissues is gingival overgrowth(GO). It is characterized by the gingival enlargement with epithelial thickening, a large number of proliferating fibroblasts and overproduction of ECM components. Fibroblast accumulation in Cs A induced GO is caused by the Inhibition of apoptosis. But CsA effect on normal human oral keratinocyte(NHOK) remains unclear. Transglutaminase 2(TGase 2) which is expressed and activated in tissue where epithelial cells undergo apoptosis has been used as a marker for apoptotic cells. The purpose of this study were to study the effect of CsA on the proliferation and apoptosis of cultured NHOK by TGase 2 expression. After primary cultured NHOK was treated by 0.1, 1.0 and 10ug/ml Cs A, growth curve, MTT assay for succinyl dehydrogenase activity and RT-PCR for TGase 2 mRNA expression were done. The obtained results were as follows. MTT assay showed about 65% cell viability at 1.0μg/ml and 40% at 10μg/ml CsA. Growth curve showed normal S curve on control & DMSO, while decreased growth rate after 3 days of higher CsA tx. TGase 2 mRNA expression of cultured NHOK was the highest at 10μg/ml Cs A. TEM showed chromosome margination, and vacuole formation and clustered mitochodria in cytoplasm of cultured NHOK after CsA tx. It suggested that higher CsA might induce apoptosis of NHOK correlated with increased TGase 2 mRNA expression

We attempted to control the maturation promoting factors (MPF) activity and nuclear remodeling of somatic cell nuclear transfer (NT) bovine embryos. Bovine ear skin fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h and activated. The nuclear remodeling type of the reconstituted embryos was evaluated 1.5 h after activation. MPF activity was assessed in enucleated and chemical treated oocytes before the injection of a donor cell. Effect of chemicals on the embryonic development was evaluated with parthenogenetic embryos. MPF activity increased significantly by caffeine treatment, but decreased by vanadate treatment (p<0.05). Caffeine or vanadate had no deleterious effect on the parthenogenetic embryo development. In caffeine treated group, premature chromosome condensation (PCC) was occurred in 72.2% of NT embryos (p<0.05). In contrast, vanadate induced the formation of a pronucleus-like structure (PN) in a high frequency (68.9%, p<0.05) without PCC (NPCC). Blastocyst development of NT embryos increased by treating with caffeine (30.3%), whereas decreased by treating with vanadate (11.4%) compared to control (22.1%, p<0.05). The results indicate that caffeine or vanadate can control of MPF activity and remodeling type of NT embryos, resulting in the increased or decreased in vitro development.

곰피추출물의 CCD-986sk cell line monolayer (human fibroblast, KCBL-21947)에 대한 피부세포 생리활성효과를 측정하고, 또한 곰피추출물의 Clone M-3 mouse melano-cyte cell line에 대한 melanin formation 저해효과를 측정하기 위해 in vitro레벨에서 실험을 실시하였다. 곰피는 다년생 갈조류의 일종으로 이 종은 한국 연안해역에서 중요한 1차생산자의 역할을 담당하고 있는

본 연구는 체세포 복제란 이식우의 분만에 있어서 혈중 스테로이드호르몬, TGF-β1 농도와 분만지연의 상관 관계를 살펴보고자 실시하였다. 대조군으로는 인공수정(AI)을 통하여 임신한 암소(cow)들을 사용하였다(AI-R). 모든 AI-R들은 자연분만(n=5, 임신 284+-0.71일)을 하였다. 분만징후를 보이지 않는 체세포 복제란 이식우(n=5, SCNT-R)들은 분만 예정일보다 10일 정도 지난 임신 292일째에 제왕절개(Caesarean section, C-sec)를 실시하여 분만하였다. 혈액 및 태반 샘플을 분만 전.후에 채취하여 형태 및 중량 등을 측정하였다. 혈장호르몬인 Progesterone(P4)와 Estradiol-17β (E2) 농도는 방사선동위원소 면역 분석 시험(RIA) 방법을 이용하여 측정하였다. 혈장 및 태반분엽의 TGF-β1 농도는 ELISA 방법으로 측정하였다. SCNT-R에서 회수한 태반의 무게는 AI-R의 것과 비교하여 유의적으로 무거웠다(p<0.05). 분만 직전 SCNT-R들의 혈장 내 P4 농도는 AI-R들의 그것과 비교하여 유의하게 높았다(p<0.01). 하지만 SCNT-R들의 혈장 내 E2 농도는 AI-R과 비교하여 상대적으로 낮게 나타났다(p<0.01). 한편, 분만 전.후 SCNT-R들에서 혈장 또는 태반분엽의 TGF-β1 단백질 발현 수준은 AI-R들과 비교하여 각각 유의적으로 높은 수준을 유지하였다(p<0.01). 이상의 결과를 종합하여 보면, 분만 시 P4 및 E2의 이상 발현과 높은 수준의 혈장 및 태반 내 TGF-β1 단백질은 체세포 복제태아의 분만지연을 야기하는 중요한 요인들 중의 하나일 것이라 사료된다.

Vaginal cytology with behavioral observation was performed in 34 estrous cycles in 16 Miniature Schnauzer dogs to evaluate the its usefulness. The mean duration of proestrus and estrus in Miniature Schnauzer based on behavioral observation and vaginal cytology was (Mean S.D., range: ) and days for proestrus, and and days for estrus, respectively. The duration of each phase of the estrous cycle was not significantly different based on between behavioral observation and vaginal cytology. The gestational length from the first day of male acceptance was days, days from the first male refusal, and days from the onset of cytologic diestrus, respectively. Vaginal cytology during the estrous cycle were significantly characteristic of large intermediate cells in proestrus, anuclear cells in estrus, small intermediate cells in diestrus, and parabasal cells and small intermediate cells in anestrus (p<0.001), respectively. Cornification index (CI) by vaginal cytology was higher in proestrus () and estrus (), then it was decreased in diestrus () and anestrus ().

조혈 줄기 세포에의 효과적인 유전자 전달은 유전자 치료의 새로운 가능성을 제시할 수 있다. 레트로바이러스를 이용한 유전자 전달 기술은 많은 기초 연구와 임상 시도가 이루어진 대표적인 바이러스이다. 그러나 현재 사용되고 있는 in vitro에서의 조혈 줄기 세포에의 유전자 도입은 조혈 줄기 세포의 분화 유도, 자기 복제 능력과homing 능력의 저하 등 많은 문제점이 있다. 본 연구는 이러한 문제점을 극복하기 위한 방법으로서 마우스의 대퇴골에 직접 레트로바이러스를 이식하는 IBM (Intra-Bone Marrow) 방법을 이용하여 조혈 줄기 세포에의 효과적인 유전자 도입을 시도하였다. IBM 이식 2주 후 마우스의 각 조직을 분석한 결과, 골수뿐 아니라 림파절, 비장, 간장 세포 등에서 유전자가 안정적으로 발현하는 것을 관찰하였다. 또한, 6.4+-2.7%의 골수조직 존재 조혈줄기/전구세포에서 도입된 유전자가 안정적으로 발현하고 있는 사실을 확인하였다. 본 연구의 결과를 바탕으로 IBM 이식 방법을 이용한 생체 조직 내 레트로바이러스의 유전자 도입은 조혈 줄기 세포를 이용한 유전자 치료에 매우 효과적인 방법이라는 사실을 시사해주고 있다.