• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Development and Reproduction
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• v.11 no.3
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• pp.187-193
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• 2007

Ethane 1,2-dimethane sulfonate(EDS), a Leydig cell specific toxicant, has been widely used to create the reversible testosterone withdrawal rat model. Though the maintenance of epididymal structure and function is highly dependent on the testosterone secreted from testis, its derivatives, dihydroxytestosterone(DHT) and estrogen, might have crucial roles. The aim of present study was to monitor the expression patterns of sex steroid receptors, cytochrome P450 aromatase(P450arom) and $5{\alpha}$-reductase in the rat epididymis up to 7 weeks after EDS injection. Adult male rats($350{\sim}400g$) were injected with a single does of EDS(75 mg/kg i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. The transcriptional activities of the target genes were evaluated by semi-quantitative RT-PCRs. The transcript level of estrogen receptor alpha($ER{\alpha}$) in EDS group was significantly higher than control level on week 1(P<0.01). After week 2, there was no significant difference in $ER{\alpha}$ levels between EDS group and control. The transcript level of estrogen receptor beta($ER{\beta}$) in EDS group was significantly higher than control level on week 1(P<0.05), lowered on weeks 2 and 3(P<0.05 and P<0.01, respectively), fluctuated during weeks 4 and 6, and elevated on week 7(P<0.05). The androgen receptor (AR) message levels increased significantly week 2(P<0.01), then returned to control level on week 3. In contrast, expression of cytochrome P450 aromatase(P450arom) decreased sharply during weeks $1{\sim}3$(P<0.01 on weeks 1 and 2; P<0.05 on week 3), then went back to control level on week 4. The mRNA level of $5{\alpha}$-reductase type 2($5{\alpha}$-RT2) increased significantly on week 4(P<0.01), then returned to control level. The present study indicated that EDS administration could induce reversible alterations in the transcriptional activities of sex steroid hormone receptors and androgenconverting enzymes in rat epididymis. EDS injection model will be useful to clarify the regulation mechanism of mammalian epididymal physiology.

• Journal of the korean academy of Pediatric Dentistry
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• v.30 no.4
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• pp.643-653
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• 2003

Fibroblast growth factor (FGF) / FGF receptor (FGFR) mediated signaling is required for skeletogenesis in cluding intramembranous and endochondral ossifications Runx2 ($Cbfa1/Pebp2{\alpha}A/AML3$) is an essential transcription factor for osteoblast differentiation and bone formation. Murine calvaria and mandible are concurrently undergoing both intramembranous bone and cartilage formations in the early developmental stage. However the mechanism by which these cartilage formations are regulated remains unclear. To elucidate the effect of FGF signaling on development of cranial sutural cartilage and Meckel's cartilage and to understand the role of Runx2 in these process, we have done both in vivo and in vitro experiments. Alcian blue staining showed that cartilage formation in sagittal suture begins from embryonic stage 16 (E16), Meckel's cartilage formation in mandible from E12. We analyzed by in situ hybridization the characteristics of cartilage cells that type II collagen, not type X collagen, was expressed in sagittal sutural cartilage and Meckel's cartilage. In addition, Runx2 was not expressed in Meckel's cartilage as well as sagittal sutural cartilage, except specific expression pattern only surrounding both cartilages. FGF signaling pathway was further examined in vitro. Beads soaked in FGF2 placed on the sagittal suture and mandible inhibited both sutural and Meckel's cartilage formations. We next examined whether Runx2 gene lies in FGF siganling pathway during regulation of cartilage formation. Beads soaked in FGF2 on sagittal suture induced Runx2 gene expression. These results suggest that FGF signaling inhibits formations of sagittal sutural and Meckel's cartilages, also propose that FGF siganling is involved in the proliferation and differentiation of chondroblasts through regulating the transcription factor Runx2.

• Journal of the korean academy of Pediatric Dentistry
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• v.32 no.3
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• pp.576-583
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• 2005

Tooth formation is a complex developmental process that is mediated through a series of reciprocal epithelial-mesenchymal interactions. Several signal pathways and transcription factors have been implicated in regulating molar crown development, but relatively little is known about the regulation of root development. It was reported that NFI-C knockout mice showed abnormal root formation with normal crown. The aims of this study are to elucidate how the NFI-C regulate the determine of root shape and odontoblasts differentiation. We carried out immunohistochemistry using cytokeratin to investigate the role of Hertwig's epithelial root sheath and DSPP mRNA in-situ hybridization to conform the nature of root dentin during root development in NFI-C knockout mice. Cytokeratin reacted with all the HERS cells and the continuity of cytokeratin positive cells between the HERS cells and enamel epithelium was lost in the cervical region both wild and K/O types. After root dentin deposition cytokeratin positive-HERS cells showed irregularity and loss of polarity in the cervical region in K/O type. DSPP mRNA was strongly expressed in odontoblasts of crown and root dentin in wild type mice, whereas expression of DSPP mRNA was restricted in odontoblast of crown dentin in the K/O type. During root formation in NFI-C knockout mice, HERS normally grow out of the crown but fail to induce odontoblast differentiation in root portion. These results suggest that NFI-C may play important roles in odontoblast differentiation during root dentin formation.

• Journal of Life Science
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• v.22 no.10
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• pp.1359-1370
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• 2012

Arabidopsis AtERF71/HRE2, a transcription activator, is located in the nucleus and is involved in the signal transduction of low oxygen and osmotic stresses. In this study, microarray analysis using AtERF71/HRE2-overexpressing transgenic plants was performed to identify genes downstream of AtERF71/HRE2. A total of 161 different genes as well as AtERF71/HRE2 showed more than a twofold higher expression in AtERF71/HRE2-overexpressing transgenic plants compared with wild-type plants. Among the 161 genes, 24 genes were transcriptional regulators, such as transcription factors and DNA-binding proteins, based on gene ontology annotations, suggesting that AtERF71/HRE2 is an upstream transcription factor that regulates the activities of various downstream genes via these transcription regulators. RT-PCR analysis of 15 genes selected out of the 161 genes showed higher expression in AtERF71/HRE2-overexpressing transgenic plants, validating the microarray data. On the basis of Genevestigator database analysis, 51 genes among the 161 genes were highly expressed under low oxygen and/or osmotic stresses. RT-PCR analysis showed that the expression levels of three genes among the selected 15 genes increased under low oxygen stress and another three genes increased under high salt stress, suggesting that these genes might be downstream genes of AtERF71/HRE2 in low oxygen or high salt stress signal transduction. Microarray analysis results indicated that AtERF71/HRE2 might also be involved in the responses to other abiotic stresses and also in the regulation of plant developmental processes.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.517-528
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• 2010

Rice seed maturation and germination involve drastic changes in water and nutrient transport, in which tonoplast aquaporins may play an important role. In the present study, gene expression profiles of 10 tonoplast intrinsic proteins (TIP) from rice were investigated by RT-PCR during seed development and germination. OsTIP3;1 and OsTIP3;2 were specifically expressed in mature seeds. Their transcript level rapidly decreased after onset of seed germination and gene expression was induced by ABA treatment. In contrast, expression of OsTIP2;1 and OsTIP4;3 was not seed specific as transcripts were found in vegetative tissues as well. Their respective transcript levels decreased at an early stage of seed development, whereas they increased at a later stage of seed germination and elongation of embryonic roots and shoots. When seed germination was inhibited by various stress conditions and ABA, expression of OsTIP2;1 and OsTIP4;3 was completely suppressed. In contrast, the expression level of OsTIP2;2 rapidly increased after seed imbibition and the transcript level was maintained under conditions inhibiting seed germination. These results implicate that tissue specific and developmental transcriptional regulation of OsTIPs in rice seeds depends on their specific function. In addition, OsTIPs can be discriminated by different potential phosphorylation and methylation sites in their protein structures. OsTIP3;1 and OsTIP3;2 possess unique phosphorylation signatures at their N-terminal domain, loop B and loop E, respectively. OsTIP2;1 and OsTIP4;3 have a potential methylation site at their Nterminal domain. This suggests that activity of specific tonoplast aquaporins may be regulated by post-translational modification as well as by transcriptional control.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.45 no.3
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• pp.217-224
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• 2019

Phthalates, endocrine disrupting chemicals, are similar in structure to sex hormones and mainly show reproductive toxicity and developmental toxicity. In this study, we analyzed 11 phthalates, including 3 kinds of phthalates prohibited in cosmetic use and 8 kinds of phthalates regulated in 'Common standards for children's products safety' and EU cosmetic regulation (EC No. 1223/2009). The phthalate analysis was optimized using GC-MS/MS. In analytical method validation, this method was satisfied in specificity, linearity, recovery rate, accuracy and MQL. Therefore, we used this method to analyze 82 products of Nail cosmetics & polish. Although six phthalates such as DBP, BBP, DEHP, DPP, DIBP and DIDP were detected at concentrations of $1.0{\sim}59.8{\mu}g/g$g, they were suitable to Korean cosmetic standards. DIBP and DBP were detected at concentration of $1.1{\sim}2.6{\mu}g/g$ in artificial nail, DBP and DEHP were $1.4{\sim}2.5{\mu}g/g$ in glue for nails, and DIBP, DBP, and DEHP were $2.5{\sim}33.3{\mu}g/g$ in nail stickers. Although substances such as DBP and DEHP in artificial nail, Glue for nails, and nail stickers were detected, they were suitable to 'Common safety standards for children's products. DIBP is not a regulated substance in Korea but showed the third highest detection rate following DBP (84.6%) and DEHP (63.4%). The concentration of phthalates detected in nail products is considered to be safe in current standards but continuous monitoring and research about non-regulated substances are also needed to be considered.

• Development and Reproduction
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• v.12 no.3
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• pp.289-296
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• 2008

BPES (Blepharophimosis/Ptosis/Epicanthus inversus Syndrome) is an autosomal dominant disorder caused by mutations in FOXL2. Affected individuals have premature ovarian failure (POF) in addition to small palpebral fissures, drooping eyelids, and broad nasal bridge. FOXL2 is a member of the forkhead family transcription factors. In FOXL2-deficient ovaries, granulosa cell differentiation dose not progress, leading to arrest of folliculogenesis and oocytes atresia. Using yeast two-hybrid screening of rat ovarian cDNA library with FOXL2 as bait, we found that small ubiquitin-related modifier (SUMO)-conjugating E2 enzyme UBE2I protein interacted with FOXL2 protein. UBE2I also known as UBC9 is an essential protein for processing SUMO modification. Sumoylation is a form of post-translational modification involved in diverse signaling pathways including the regulation of transcriptional activities of many transcriptional factors. In the present study, we confirmed the protein-protein interaction between FOXL2 and UBE2I in human cells, 293T, by in vivo immunoprecipitation. In addition, we generated truncated FOXL2 mutants and identified the region of FOXL2 required for its association with UBE2I using yeast-two hybrid system. Therefore, the identification of UBE2I as an interacting protein of FOXL2 further suggests a presence of novel regulatory mechanism of FOXL2 by sumoylation.

• Molecules and Cells
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• v.45 no.12
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• pp.896-910
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• 2022

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and potentially fatal virus. So far, most comprehensive analyses encompassing clinical and transcriptional manifestation have concentrated on the lungs. Here, we confirmed evident signs of viral infection in the lungs and spleen of SARS-CoV-2-infected K18-hACE2 mice, which replicate the phenotype and infection symptoms in hospitalized humans. Seven days post viral detection in organs, infected mice showed decreased vital signs, leading to death. Bronchopneumonia due to infiltration of leukocytes in the lungs and reduction in the spleen lymphocyte region were observed. Transcriptome profiling implicated the meticulous regulation of distress and recovery from cytokine-mediated immunity by distinct immune cell types in a time-dependent manner. In lungs, the chemokine-driven response to viral invasion was highly elevated at 2 days post infection (dpi). In late infection, diseased lungs, post the innate immune process, showed recovery signs. The spleen established an even more immediate line of defense than the lungs, and the cytokine expression profile dropped at 7 dpi. At 5 dpi, spleen samples diverged into two distinct groups with different transcriptome profile and pathophysiology. Inhibition of consecutive host cell viral entry and massive immunoglobulin production and proteolysis inhibition seemed that one group endeavored to survive, while the other group struggled with developmental regeneration against consistent viral intrusion through the replication cycle. Our results may contribute to improved understanding of the longitudinal response to viral infection and development of potential therapeutics for hospitalized patients affected by SARS-CoV-2.