E3 SUMO ligase AtSIZ1 regulates FLC-mediated flowering repression

학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학과, 2014. 8. 서학수.Flowering Locus C (FLC), a floral repressor, is antagonistic regulator for transition from vegetative to the reproductive phase. FLC represses expression of several genes involved in floral induction and its transcription is positively or negatively regulated by FRIGIDA or by vernalization, respectively. Although several factors affecting the transcription of FLC have been described, the post-translational regulation of FLC stability and function has not been clearly characterized. Here, I investigated the mechanisms regulating the activity and stability of the FLC protein. Sumoylation, a post translational process of attaching small ubiquitin-related modifier (SUMO) to a lysine residue in proteins through an enzyme cascade catalyzed by E1, E2 and E3 enzymes, plays crucial role in protein stability and biological processes. Bimolecular fluorescence complementation, co-immunoprecipitation and in vitro pull down analysis showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro sumoylation assays showed that FLC is modified by SUMO in presence of SUMO-activating enzyme E1 and conjugating enzyme E2 but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of sumoylation site)-overexpressing plants was comparable to that in wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. These results indicate that the sumoylation of FLC is critical for its role in the control flowering time and AtSIZ1 positively regulates FLC-mediated floral suppression.ABSTRACT Ⅰ CONTENTS Ⅲ LIST OF FIGURES Ⅵ LIST OF TABLES Ⅷ LIST OF ABBREVIATIONS Ⅸ LITERATURAL REVIEWS 1 1. SUMOylation 1 1.1. The SUMOylation pathway 1 1.2. Components of the SUMO pathway in Arabidopsis 3 2. Role of the SUMO in plants 6 2.1. Responses to abiotic stress 6 2.2. Responses to hormone signaling 9 2.3 Control of flowering 10 2.4. Defense reactions to pathogen attack 11 2.5. Metabolic regulation 12 3. Regulation of flowering time in Arabidopsis 14 3.1. Photoperiod pathway 14 3.2. Gibberellin pathway 15 3.3. Autonomous pathway 16 3.4. vernalization pathway 18 INTRODUCTION 20 MATERIALS AND METHODS 22 1. Plant materials and growth conditions 22 2. Construction of recombinant plasmids 22 3. Production of transgenic Arabidopsis plants 24 4. Purification of recombinant proteins 24 5. In vitro binding assay 25 6. In vivo Co-immunoprecipitation 27 7. Sumoylation assay 27 8. BiFC assay 28 9. Quantitative Real-Time RT-PCR analysis 29 10.β-estradiol and cycloheximide treatments 30 11. Cell-free degradation assay 30 12. Yeast two-hybrid assays 31 13. Investigation of flowering time 31 RESULTS 35 1. AtSIZ1 physically interacts with FLC 35 2. FLC sumoylated without AtSIZ1 41 3. AtSIZ1 inhibits FLC sumoylation 44 4. Identification of sumoylation site on FLC 46 5. FLC stabilized by AtSIZ1 49 6. Modification by SUMO is necessary for flowering repression 57 7. Mutant FLC interacts with AtSIZ1 and FLC 62 DISCUSSION 65 REFERENCES 71 ABSTRACT IN KOREAN 90Docto

A case of pulmonary sequestration mimicking mediastinal mass detected by prenatal ultrasonography

Pulmonary sequestration is a developmental anomaly of broncho-pulmonary foregut with nonfunctioning parenchymal tissue, which usually supplied by systemic circulation. Pulmonary sequestration is detected by ultrasonography as a homogeneous echogenic mass and also by Doppler blood flow from systemic circulation to the mass. Pulmonary sequestration is classified into intralobar type and extralobar type. Extralobar type accounts for only 15~25% of the cases and it is subdivided into intrathoracic forms, which are most commonly found and extrathoracic type, which includes intraabdominal, retroperitoneal, or mediastinal masses. We report a rare case of prenatal detection of mediastinal mass with a brief review of literatures, which was confirmed to be a pulmonary sequestration by surgical mass excision after birth.ope

Surgical treatment of pulmonary embolism after cesarean section

Pulmonary embolism (PE) is the leading cause of maternal deaths and the incidence has been increased in recent years in Asian countries. Although the treatment options available for patients with massive PE include thrombolytic therapy, catheter-directed thrombectomy, and surgical embolectomy, there are no conclusive data or evidence on the appropriate treatment of PE. We experienced a case of massive PE with large thrombus involving major pulmonary arteries immediately after emergency cesarean section and successfully treated by thrombectomy, so hereby report the case.ope

A case of round ligament varicosities during pregnancy

Estimated incidence of round ligament varicosities in pregnancy is not known and often times it is confused with inguinal hernia due to their clinical similarities. When a patient is presented with inguinal mass especially in association with varicosity in the genital region or lower extremity, round ligament varicosity must be considered as a plausible diagnosis. Depiction of "bag of worms" on color Doppler ultrasonography is diagnostic of the round ligament varicosity and it is known to resolve spontaneously following delivery. We report a case of round ligament varicosities that was diagnosed at 29 weeks of gestation with a brief review of the literaturesope

Random urine protein/creatinine ratio readily predicts proteinuria in preeclampsia

OBJECTIVE: To assess the diagnostic accuracy of random urine protein-creatinine (P/C) ratio for prediction of significant proteinuria in preeclampsia as an alternative to the time-consuming 24-hour urine protein collection. METHODS: Retrospective record analysis was performed on 140 pregnant women who were admitted with suspicion for preeclampsia from January 2006 to June 2011. Random urine protein and/or 24-hour urine protein levels were assessed and their correlation to random urine P/C ratio and 24-hour urine protein excretion was evaluated. RESULTS: Out of 140 patients, random urine P/C ratio or/and 24-hour urine protein was performed in 79 patients to evaluate significant proteinuria. Of 79 patients, 46 (58%) underwent both tests whereas in 33 women (42%) 24-hour urine collection was not available due to urgent delivery. In 39 cases (85%), significant proteinuria (≥300 mg/24 hr) was detected with 6 cases (13%) having values over 5,000 mg/24 hr, corresponding to the diagnosis of severe preeclampsia. Random urine P/C ratio highly correlated with 24-hour urine protein excretion (r=0.823, P<0.01). The optimal random urine P/C ratio cutoff points were 0.63 and 4.68 for 300 mg/24 hr and 5,000 mg/24 hr of protein excretion, respectively. with each sensitivity, specificity, and positive and negative predictive values of 87.1%, 100%, 100%, and 58.3%; and 100%, 85%, 50%, and 100%, for significant and severe preeclampsia, respectively. CONCLUSION: Random urine P/C ratio is a reliable indicator of significant proteinuria in preeclampsia and may be better at providing earlier diagnostic information than the 24-hour urine protein excretion with more accuracy than the urinary dipstick test.ope

Frontomaxillary facial angle measurements in euploid Korean fetuses at 11 weeks' to 13 weeks 6 days' gestation.

OBJECTIVE: The purpose of this study was to evaluate the distribution of fetal frontomaxillary facial angles in a euploid Korean population at 11 weeks' to 13 weeks 6 days' gestation. METHODS: Three-dimensional volumes of the fetal head were obtained from women with low-risk singleton pregnancies at 11 weeks' to 13 weeks 6 days' gestation who consented to this prospective study. Only fetuses with either a normal karyotype confirmed by amniocentesis or no abnormalities after delivery were considered eligible for analysis and were characterized as euploid for the purposes of this study. Women with multiple pregnancies and those who were lost to follow-up and fetuses with abnormal karyotypes or anomalies diagnosed in utero or postnatally were excluded. The frontomaxillary facial angle was measured twice offline by a single examiner. Cases were categorized by crown-rump length (CRL) in 10-mm intervals for analysis of the frontomaxillary facial angle. RESULTS: Among 375 enrolled cases, 158 were eligible for frontomaxillary facial angle analysis. The overall mean frontomaxillary facial angle ± SD was 88.6° ± 9.7°. The mean frontomaxillary facial angle for fetuses with a CRL of 40 to 49 mm (n = 35) was 93.7°; 50 to 59 mm (n = 53), 92.6°; 60 to 69 mm (n = 36), 85.3°; and 70 to 79 mm (n = 34), 81.0°, showing an inverse relationship between the mean frontomaxillary facial angle and CRL (r = -0.5334; P < .0001). The proportion of cases with frontomaxillary facial angles of 85° or greater was 60.8%, and that of cases with angles of 90° or greater was 37.3%. CONCLUSIONS: Ethnic differences in frontomaxillary facial angle measurements should be considered when incorporating the frontomaxillary facial angle in fetal aneuploidy screening in the Korean population.ope

First Trimester Placental Volume and Second Trimester Uterine Artery Doppler Velocimetry in the Prediction of Perinatal Outcomes

Objective: To determine to what extent the placental quotient (PQ: placental volume/crown-rump length) is able to detect adverse perinatal outcomes and to compare the value of first trimester placental volume and second trimester uterine artery Doppler velocimetry for predicting adverse perinatal outcomes. Methods: This was a prospective study comprising of 263 women with singleton pregnancies attending our hospital for nuchal translucency screening at 10-13 weeks of gestation. Three dimensional ultrasound was used to obtain images for measurement of placenta volume at 10-13 weeks of gestation. In addition, Doppler assessment of both uterine arteries was carried out for measurement of the pulsatility index (PI) in the second trimester and the mean PI of the two vessels was calculated. The variables of adverse perinatal outcomes were preeclampsia, preterm delivery, small for gestational age (SGA), 5-minute APGAR score, and admission to the neonatal intensive care unit. Results: Of the initial 263 pregnancies originally participating, 219 women who delivered at our institution were included in the final analysis. Pregnancy complications occurred in 27 (12.3%) of the 219 pregnancies. Comparison between crownrump length and placental volume proved a significant correlation (r=0.474, p<0.001). There were no correlation between PQ and uterine artery Doppler velocimetry PI. PQ was significantly lower in SGA group and neonatal intensive care unit admission group (p=0.049, p=0.019, respectively). Uterine artery Doppler velocimetry PI was significantly higher in SGA group (p=0.024). PQ in the first trimester and uterine artery Doppler velocimetry in the second trimester had similar sensitivities for predicting SGA. Conclusions: PQ in the first trimester and uterine artery Doppler velocimetry PI in the second trimester have significant difference in SGA group compared to appropriate for gestational age, and have similar sensitivities for predicting SGA. While both methods seem to be insufficient for screening, the PQ method has the potential advantage of being performed in the first trimesterope

융모양막염이 있는 조산환자에서 microRNA-548에 의한 high mobility group box 1 발현 조절에 대한 연구

Infections and inflammations of the amniotic cavity are often accompanied by histologic chorioamnionitis, which is associated with both preterm delivery and adverse perinatal outcome. High mobility group box 1 (HMGB1) is a prototypic alarmin. It plays an important role in the pathogenesis of inflammatory process in pregnant women, and is associated with spontaneous preterm birth. This study was conducted to compare the levels of HMGB1 in amniotic fluid and amnion membranes in women with chorioamnionitis/intra-amniotic inflammation to those in healthy controls. We also aimed to determine the expression of MicroRNA-548 (miR-548) cluster in amnion membrane, and to elucidate its involvement in regulating HMGB1 expression and its function in human amniotic epithelial cells (hAECs). Levels of HMGB1 in amniotic fluid (AF) were compared between women with intra-amniotic inflammation (n=34) and normal controls (n=14). We also determined HMGB1 expression levels in amnion membranes of women with chorioamnionitis (n=6) against those in healthy controls (n=4). MiR-548 cluster was predicted to bind HMGB1 by a bioinformatics analysis. To investigate the causal relationship between miR-548 cluster and HMGB1, a repressed and forced expression assay in hAECs was performed. The levels AF HMGB1 were significantly higher in patients with intra-amniotic inflammation than in those without inflammation. HMGB1 expression was also increased in amnion membranes from women with preterm birth and chorioamnionitis as compared to normal controls. MiR-548 cluster was significantly under-expressed in amnion membranes from patients with chorioamnionitis than in normal term controls. Repressed expression of miR-548 up-regulated HMGB1 expression in hAECs and increased its release from hAECs. Moreover, forced expression of miR-548 suppressed HMGB1 and inflammatory cytokines in hAECs, which increased when treated with lipopolysaccharide. In conclusion, intra-amniotic inflammation suppresses miR-548, which increases HMGB1 expression in the amnion membrane and HMGB1 release. Moreover, miR-548 can alter the inflammatory responses in hAECs. MiR-548 might be involved in the pathogenesis of preterm birth and chorioamnionitis by regulating HMGB1. 자궁내감염 및 염증은 융모양막염에서 흔히 동반되며 이는 자연조산 및 불량한 임신 예후와 연관되어 있다. High mobility group box 1 (HMGB1)은 원형위험신호물질 (prototypic alarmin)로서 임신 중 염증반응에 관여하여 조산발생에 중요한 역할을 한다고 알려져 있다. 이 연구는 융모양막염 및 자궁내염증이 있는 환자군과 정상군사이에 HMGB1의 발현 정도를 비교하고 microRNA-548이 HMGB1의 발현조절에 어떠한 역할을 하는지 알아보고자 시행되었다. 자궁내염증이 있는 환자 34명과 정상군 14명을 대상으로 자궁내 HMGB1의 발현 정도를 비교하였을 때 자궁내염증이 있는 환자군에서 유의하게 HMGB1의 발현이 높았다. 다음으로 자궁강을 둘러싸고 있는 태아막인 양막에서 HMGB1의 발현 정도를 비교하였을 때 융모양막염이 있는 환자의 양막에서 정상군과 비교하여 유의하게 HMGB1의 mRNA 및 단백질의 발현이 높게 나타났다. 또한 microRNA-548의 발현을 살펴보았을 때 융모양막염이 있는 환자군의 양막에서 정상군에 비하여 microRNA-548의 발현이 현저하게 낮았다. 다음으로 microRNA-548과 HMGB1의 상호관계를 규명하기 위하여 양막에서 양막상피세포를 분리 및 배양하여 실험을 진행하였다. 먼저 MicroRNA-548 inhibitor를 양막상피세포에 트랜스펙션하여 microRNA-548의 발현을 억제시켰을 때 양막상피세포에서 HMGB1의 발현이 유의하게 증가하였으며 양막상피세포에서 분비되는 HMGB1의 양도 증가하였다. 또한 양막상피세포를 배양하는 배지에 lipopolysaccharide를 처리하여 염증반응을 유발시켰을 때 MicroRNA-548 mimic을 트랜스펙션시킨 양막상피세포에서는 HMGB1의 발현 및 유리가 증가하지 않았다. 또한 microRNA-548 mimic을 트랜스펙션시킨 양막상피세포에서는 lipopolysaccharide로 염증반응을 유발시켰을 때 염증싸이토카인 및 케모카인 유리가 증가하지 않았다. 결론적으로 자궁내염증은 양막에서 microRNA-548을 억제시키고 이는 양막상피세포에서 HMGB1의 발현과 유리를 증가시킨다. 또한 microRNA-548은 양막상피세포에서 염증반응을 조절할 수 있다. 따라서 microRNA-548은 HMGB1의 발현을 조절함으로써 융모양막염이 있는 산모에서 조산을 유발하는데 역할을 한다.open박

HPV 4가 백신에서 변형된 lipopolysaccharide/bacterial DNA fragments/aluminium salt 복합체가 보조제로서 면역 반응에 미치는 영향

Dept. of Medicine/석사[한글]백신 보조제는 면역 반응을 향상시키고 백신에 사용되는 항원의 양을 줄이는데 도움이 되는 물질로 보다 효과적이고 안전한 보조제 (adjuvant) 를 개발하는 것은 백신 연구에서 중요하고 어려운 과제이다. E.coli DNA의 일부분과 변형된 lipopolysaccahride (LPS)의 복합체를 CIA07이라고 명명하였을 때 본 연구에서는 HPV 4가 백신에서 보조제로 aluminium salt만을 사용하였을 때에 비하여 CIA07을 첨가하였을 때의 효과를 알아보고자 한다. 쥐를 5마리씩 5군으로 하여 각각 다음과 같이 3주 간격으로 2회 근육주사 하였다. 1군은 대조군으로 생리식염수, 2군은 HPV 4가 백신, 3군은 HPV 4가 백신과 세균 DNA fragments, 4군은 HPV 4가 백신과 변형된 LPS, 5군은 HPV 4가 백신과 CIA07을 근육주사 하였고 두 번째 백신 주사 후 1주와 4주 후에 면역반응을 측정하였다. 보조제로 변형된 LPS와 CIA07을 첨가한 경우 aluminium salt만을 사용한 경우보다 HPV 16/18 L1 VLPs에 대한 항체 (IgG)가 유의하게 증가하였다. 또한 HPV 16/18 L1 VLPs specific IgG isotype을 측정하였을 때 CIA07을 보조제로 첨가하였을 경우 aluminium salt만을 사용한 경우보다 두 번째 백신 주사 4주 후 측정한 HPV 16/18 L1 VLPs에 대한 IgG1 와 IgG2a 항체는 모두 증가하였다. 그러나 변형된 LPS를 보조제로 사용한 경우 IgG1은 효과적으로 증가시키는 반면 IgG2a는 증가시키지 못하였다. HPV 16/18 L1 VLPs specific IgG2a/IgG1 ratio을 계산한 결과 두 번째 백신 주사 4주 후에, CIA07을 보조제로 첨가한 경우 aluminium salt만을 사용하였을 경우보다 높은 수치를 보여 CIA07을 첨가한 보조제를 사용하였을 경우 Th1형 면역반응을 더 효과적으로 유도할 수 있다는 것을 시사하였다. 이번 연구 결과를 종합하여 볼 때 CIA07을 HPV 4가 백신의 보조제로 첨가할 경우 aluminium salt만을 사용하였을 경우보다 체액성 및 세포성 면역 반응을 보다 향상시킬 수 있어 효과적인 면역 보조제로서의 역할을 할 수 있으리라 기대된다. [영문]Developing efficient and safe adjuvants for use in human vaccines remains both a challenge and a necessity. CIA07 is a immunostimulatory agent composed of E.coli DNA fragments and modified lipopolysaccharide (LPS) lacking the lipid A moiety. In this study, we investigated adjuvant activity of CIA07 using HPV L1 VLPs as the immunogen. Mice were immunized intramuscularly two times at 3-week intervals with HPV 6/11/16/18 L1 VLP vaccine alone or in combination with bacterial DNA fragments, modified LPS or CIA07, and immune responses were assessed. Modified LPS and CIA07 adjuvanted formulation showed significantly higher titers of HPV 16/18 L1 VLPs specific antibodies than aluminium salt. HPV 16/18 L1 VLPs specific IgG isotype titers were measured. Modified LPS adjuvanted formulation stimulated the IgG1 antibody response effectively, but did not induce a significant increase in the IgG2a antibody response. On the other hand, CIA07 adjuvanted formulation induced significantly higher titers of HPV 16/18 L1 VLPs specific IgG1 as well as IgG2a antibodies at 28-day post-immunization II. Furthermore, the ratio of IgG2a to IgG1 antibody titers in mice administered with CIA07 adjuvanted formulation was higher compared to aluminium salt at 28-day post-immunization II, indicating that CIA07 could stimulate higher Th1-type immune responses to HPV 16/18 L1 VLPs. These data demonstrated that CIA07 adjuvanted formulation has the ability to induce higher humoral and cellular immune responses in mice when compared with the aluminium salt, and support the role of the CIA07 as an effective adjuvant to the HPV quadrivalent recombinant vaccine.ope

A nontoxic derivative of lipopolysaccharide increases immune responses to Gardasil HPV vaccine in mice

Human papillomavirus (HPV) is the causative agent of cervical cancer, the second most common cause of cancer death in women worldwide. The licensed HPV vaccine Gardasil((R)) from Merck & Co. is a quadrivalent vaccine containing virus-like particles (VLPs) of the L1 proteins from HPV types 6, 11, 16, and 18 adsorbed on aluminum salts (alum). CIA07 is an immunostimulatory agent comprised of bacterial DNA fragments (CIA02) and a nontoxic derivative of lipopolysaccharide (CIA05) that has been shown to have antitumor activity and adjuvant activity for viral and bacterial vaccine antigens. We investigated whether these CIAs are capable of promoting the immune response to Gardasil. Balb/c mice were immunized intramuscularly twice three weeks apart with 1/20 human dose of Gardasil alone or in combination with CIA02, CIA05 or both, and immune responses were assessed. The serum anti-HPV16 L1 VLP IgG antibody titer was significantly higher in mice administered CIA05 or CIA05 plus CIA02, but not in those given CIA02, compared with mice given Gardasil alone. A secreted alkaline phosphatase (SEAP)-based pseudovirus neutralization assay showed increased neutralizing antibody titers in both CIA05 and CIA05 plus CIA02 groups. Coadministration of CIA05 with Gardasil led to a marked increase in serum IgG2a antibody titer and the percentage of interferon (IFN)-gamma(+) cells in the spleen, indicating that CIA05 effectively promotes Th1-type immune responses. These data indicate that CIA05, in synergy with alum, enhances the immune response to HPV L1 VLPs and suggest its potential as an adjuvant for the development of a potent prophylactic HPV vaccine.ope