알루미나 튜브 지지체의 표면에 NaA제올라이트 분리막을 수열합성법으로 제조하고, 이를 투과증발 공정에 사용하였다. 분리막의 제조에 사용된 지지체는 기공율 40%, 기공경 0.7 μm를 갖는 지지체를 사용하였으며, 수열합성법으로 제 조된 NaA제올라이트 분리막은 약 18 μm의 두께를 나타내었다. 제조된 분리막 은 단일 분리막 형태와 분리막 모듈로 구성하여 각각 투과증발 성능을 평가하였으며, 단일 분리막은 10L Batch type반응기에서 투과증발 공정을 수행하고, 분리막 모듈은 1 ton Batch type 반응기에서 투과증발 공정을 수행하여 그 성능을 평가하였다.

To understand molecular mechanisms underlying adaptation of plant cells to saline stress and stress memory, we developed Arabidopsis callus suspension-cultured cells adapted to high salt. Adapted cells to high salt exhibited enhanced tolerance compared to control cells. Moreover, the salt tolerance of adapted cells was stably maintained even after the stress is relieved, indicating that the salt tolerance of adapted cells was memorized. Salt-adapted and stress memorized cells were densely aggregated and formed multi-layered cell lump. Cell morphology analysis using transmission electron microscopy indicated that cell wall thickness of salt-adapted cells was significantly induced compared to control cells. In order to characterize metabolic responses of plant cells during adaptation to high salt stress as well as stress memory, we compared metabolic profiles of salt-adapted and stress-memorized cells with control cells by using NMR spectroscopy. A principle component analysis showed clear metabolic discrimination among control, salt-adapted and stress-memorized cells. Compared with control cells, metabolites related to shikimate metabolism such as tyrosine, and flavonol glycosides, which are related to protective mechanism of plant against stresses were largely up-regulated in adapted cell lines. Moreover, coniferin, a precursor of lignin, was more abundant in salt-adapted cells than control cells. The results provide new insight into metabolic level mechanisms of plant adaptation to saline stress as well as stress memory.

To identify novel signaling components involved in regulation of plant responses to phosphate (Pi) starvation, we screened an Arabidopsis T-DNA activation tagging library for mutants with altered Pi-starvation responses. Here, we report the identification and characterization of novel activation-tagged mutant involved in Pi starvation signaling in Arabidopsis. The hpd (hypersensitive to Pi deficiency) mutant exhibits enhanced phosphate uptake and altered root architectural change under Pi starvation compared to wild type. Expression analysis of auxin-responsive DR5::GUS reporter gene in hpd mutant indicated that both auxin biosynthesis and auxin translocation under Pi starvation are suppressed in hpd mutant plants. Impaired auxin translocation in roots of hpd mutant was attributable to abnormal root architecture changes in Pi starvation conditions. Mis-regulation of auxin translocation in hpd mutant was further confirmed by analysis of expression patterns of auxin efflux carrier proteins, PIN-FORMED (PIN) 1, 2, and 3 fused with GFP. Not only expression levels but also expression domains of PIN proteins were altered in hpd mutant in response to Pi starvation. Molecular genetic analysis of hpd mutant revealed that the mutant phenotype is caused by the lesion in ENHANCED SILENCING PHENOTYPE4 (ESP4) gene whose function is proposed in mRNA 3’-end processing. The results propose that mRNA processing plays crucial roles in Pi homeostasis as well as developmental reprograming in response to Pi deprivation in Arabidopsis.

The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages.

The aim of this study was to analyze the effect of antifreeze proteins (AFPs) on vitrification of mouse mature (MII) oocytes. We studied about 3 types of AFPs from different origins (FfIBP, LeIBPand Type III AFP). The MII oocytes were obtained from 4-week-old BD-F1 mice. Vitrification of oocyte was performed by 2 steps using the Cryotop (equilibration: 7.5% EG + 7.5% PROH for 5 min, vitrification: 15% EG + 15% PROH + 0.5M sucrose for 1 min). The concentrations of AFPs added to these solutions were 0.05 mg/ml for FfIBP and 0.1 mg/ml for LeIBP and Type III AFP. After fertilization, embryo development was assessed up to 5 days. Through immunostaining of vitrified-warmed oocytes, we assessed the normal meiotic spindle. Also, intracellular ROS and mitochondrial activity was analyzed. In the developmental stages, FfIBP and LeIBP groups showed significantly higher survival rates. In the blastocyst and apoptotic blastomere rates were significant differences in AFPs treated groups. AFPs treated groups were significantly higher in blastocyst cell numbers than control group. Among the AFPs treated groups, FfIBP, LeIBP groups were significantly higher rates. And, in cleavage rates, FfIBP group was significantly higher rates than the other groups. In vitrified-warmed MII oocytes, the normal meiotic spindle organization and chromosome alignment rate was significantly higher in FfIBP and LeIBP groups. And in intracellular ROS levels, control group was significantly increased than AFPs treated groups. However, in the mitochondrial activity, LeIBP group was significantly higher than control, FfIBP and LeIBP groups. AFPs treated groups were significant differences in development, meiotic spindle organization and intracellular ROS levels. And in the AFPs treated groups, FfIBP and LeIBP groups were significantly higher rates in normal meiotic spindle and mitochondrial activity than Type III AFP group respectively. In conclusion, FfIBP and LeIBP can be thought to improve oocyte cryopreservation efficiency.

Ovarian tissue cryopreservation and transplantation causes follicle depletion. To overcome this problem, we investigate the effect of Anti-Müllerian hormone (AMH), a follicle recruitment control hormone, supplementation before and/or after mouse ovarian transplantation. A total of 120 5-week-aged BD F-1 female mice were used. The mice were randomly divided into four groups according to AMH doses (0, 5, 25, 125 μg/mL, respectively). AMH was injected intraperitoneally on every other day for a week before, after, or before and after transplantation of ovaries under kidney capsules was performed. One week after transplantation, follicular normality was evaluated by histological analysis and TUNEL assay. In Group A and C, morphologically intact follicle (G1) ratios of AMH treated groups showed no statistically significant difference. In Group B, G1 ratios of 25 and 125 μg/mL of AMH treated groups were higher than those of 5 μg/mL treated group, but there was no improvement in G1 ratio after AMH treatment. In every group, apoptotic follicle ratios did not show any trend according to AMH treatment. Proportions of primordial follicle were not significantly different according to AMH treatment in all groups. The result of the present study demonstrated that AMH treatment during on transplantation of cryopreserved ovaries has no significant effect on follicle survival and prevention of follicle depletion.

Objective : To investigate the effects of Simvastatin and Methylprednisolone on ovarian tissue cryopreservation and transplantation using mouse models. Methods : The mice were randomly distributed into 1 control and 3 experimental groups. The B6D2F1 mice were given oral Simvastatin (5 mg/kg), intravenous Methylprednisolone (15 mg/kg), or a combination of both at 2 hours before ovariectomy. Same volume of normal saline was given perorally in the control group at 2 hours before ovariectomy. The ovarian tissues were vitrified accrording to our protocols. The vitrified ovaries were warmed 1 week later and auto-transplanted under bilateral kidney capsules. The ovaries and blood sera were collected at 2, 7 or 21 days after transplantation. Histological analysis, TUNEL assay, immuno-histochemistry for CD31, serum AMH level and embryonic development after in vitro fertilization were assessed for evaluation. Results : With regard to the total grade 1 follicle rate, both Simvastatin or Methylprednisolone treated groups were significantly increased at 2, 7 or 21 days after transplantation (except Simvastatin treated group at 7 days). A combination of Simvastatin and Methylprednisolone group was significantly improved in terms of the total G1 follicle rate, apoptotic follicle rate, CD31 positive area and serum AMH after ovarian tissue transplantation. However, there were no statistically difference with respect to the oocyte maturation rate, blastulation rate, and the other embryonic development parameters after in vitro fertilization procedure among the four groups. Conclusion : Our results suggest that combined donor Simvastatin and Methylprednisolone have beneficial effects on the quality and function of transplanted ovarian tissues.

Study question: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? Summary answer: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. What is known already: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified–.warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. Study design, size, duration: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. Participants/materials, setting, methods: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of a-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–.warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. Main results and the role of chance: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–.58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–.92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. Limitations, reasons for caution: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. Wider implications of the findings: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

The innate immune system is the only defense weapon that invertebrates have, and it is the fundamental defense mechanism for fish. The innate immune response is important in newly hatched flounders because it is closely involved in the initial feeding phase, which is why it is essential for survival during the juvenile period. The expression analysis of genes involved in the innate immune response in the olive flounder (Paralichthys olivaceus) in the days after hatching is incomplete. Therefore, we have begun to examine the expression patterns of genes specifically induced during the development of the innate immune system in newly hatched flounders. Microscopic observation showed that pronephron formation corresponded with the expression of perforin-encoding gene. These results suggest that perforin plays a vital role in the innate immunity of the kidney during developmental stages. Perforin expression was strong at the start of the development of the innate immune response, and continued throughout all the development stages. Our findings have important implications with respect to perforin’s biological role and the evolution of the first defense mechanisms in olive flounder. Further studies are required to elucidate the perforin-mediated innate immunity response and to decipher the functional role of perforin in developmental stages.

Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.

Phosphorus is one of the macronutrients essential for plant growth and development, as well as crop productivity. Many soils around the world are deficient in phosphate (Pi) that plants can utilize. To cope with the stress of Pi starvation, plants have evolved many adaptive strategies, such as changes of root architecture and enhanced Pi acquisition form soil. To understand molecular mechanism underlying Pi starvation stress signaling, we characterized the activation-tagged mutant showing altered responses to Pi deficiency compared to wild type Arabidopsis and named hsp3 (hypersensitive to Pi starvation3). hsp3 mutant exhibits enhanced phosphate transporter activity, resulting in higher Pi content than wild type. However, in root architectural change under Pi starvation, hsp3 shows hyposensitive responses than wild type, such as longer primary root elongation, lower lateral root density. Histochemical analysis using hsp3 mutant expressing auxin-responsive DR5::GUS reporter gene, indicated that auxin allocation from primary to lateral roots under Pi starvation is aborted in hsp3 mutant. Molecular genetic analysis of hsp3 mutant revealed that the mutant phenotype is caused by the lesion in ENHANCED SILENCING PHENOTYPE4 (ESP4) gene whose function is proposed in mRNA 3’ end processing. Here, we propose that mRNA processing plays a crucial role in Pi homeostasis in Arabidopsis.

In order to adapt to various environmental stresses, plants have employed diverse regulatory mechanisms of gene expression. Epigenetic changes, such as DNA methylation and histone modifications play an important role in gene expression regulation under stress condition. It has been known that some of epigenetic modifications are stably inherited after mitotic and meiotic cell divisions, which is known as stress memory. To understand molecular mechanisms underlying stress memory mediated by epigenetic modifications, we developed Arabidopsis suspension-cultured cell lines adapted to high salt by stepwise increases in the NaCl concentration up to 120 mM. Adapted cell line to 120 mM NaCl, named A120, exhibited enhanced salt tolerance compared to unadapted control cells (A0). Moreover, the salt tolerance of A120 cell line was stably maintained even in the absence of added NaCl, indicating that the salt tolerance of A120 cell line was memorized even after the stress is relieved. By using salt adapted and stress memorized cell lines, we intend to analyze the changes of DNA methylation, histone modification, transcriptome, and proteome to understand molecular mechanisms underlying stress adaptation as well as stress memory in plants.