• Journal of dental hygiene science
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• v.13 no.3
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• pp.321-329
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• 2013

Although it has been reported that lysyl oxidase (LOX) is involved in odontoblastic differentiation, the role of LOX on odontoblastic differentiation by hydrogen peroxide ($H_2O_2$) have not been clarified. In the present study, we investigated whether $H_2O_2$, reactive oxygen species (ROS), is modulated the messenger RNA (mRNA) expression and activity of LOX during odontoblastic differentiation of human dental pulp (HDP) cells. The mRNA expression was quantified by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, and LOX enzyme activity was measured by high sensitive fluorescent assay. Expression of the odontoblastic differentiation marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX and LOXL. The $H_2O_2$-induced mRNA expression of LOX family was significant reduction of LOX, LOXL, and LOXL3 mRNA levels in HDP cells. LOX enzyme activity was increased at $H_2O_2$ 0.3 mM for 24 hours. The mRNA expression of alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) was inhibited by LOX- and LOXL-specific siRNAs whereas the mRNA expression of dentin matrix protein1 (DMP1), and dentin sialophosphoprotein (DSPP) was inhibited by LOX-specific siRNA. In LOX enzyme activity, siRNA-induced knockdown of both LOX and LOXL inhibited the total amine oxidase activity in HDP cells, as in the case of mRNA expression. In conclusion, the essential role of $H_2O_2$ on odontoblastic differentiation suggests that its regulation by LOX may have pharmacologic importance in HDP cells.

• Journal of Pharmaceutical Investigation
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• v.25 no.4
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• pp.299-305
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• 1995

Cloning the gene encoding a dipeptide transporter is necessary for understanding the absorption mechanism of peptides and peptide-like drugs in the gastrointestinal tract. Functional expression of a dipeptide transporter after microinjection into Xenopus laevis oocytes was performed using the mRNA purified from human intestinal HT-29 cells. Fifty nanoliters of purified mRNA (1 mg/mL) were microinjected into healthy oocytes followed by incubation for 4 days in order to express a dipeptide transporter. Functional expression was determined by a uptake assay using 10 Ci/mL $[^3H]-glycylsarcosine$, a dipeptide substate of the transporter. Seasonal variability and batch-to-batch variability were greater in summer. The usage of beveled micropipettes improves viability of oocytes at 4 days after microinjection. Expression of a dipeptide transporter in oocytes after microinjection of mRNA obtained from HT-29 cells was significantly larger than those after microinjection of water or mRNA obtained from the rabbit intestine.

• Animal cells and systems
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• v.5 no.3
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• pp.217-224
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• 2001

The present study examines the expression and regulation of gonadotropin-releasing hormone (GnRH) and its receptor (GnRH-R) mRNA levels during mouse ovarian development. A fully processed, mature GnRH mRNA together with intron-containing primary transcripts was expressed in the immature mouse ovary as determined by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). The size of ovarian GnRH mRNA was similar to that of hypothalamus, but its amount was much lower than that in the hypothalamus. Quantitative RT-PCR procedure also revealed the expression of GnRH-R mRNA in the ovary, but the estimated amount was a thousand-fold lower than that in the pituitary gland. We also examined the regulation of ovarian GnRH and GnRH-R mRNA levels during the follicular development induced by pregnant mare's serum gonadotropin (PMSG) and/or human chorionic gonadotropin (hCG). Ovarian luteinizing hormone receptor (LH-R) mRNA was abruptly increased st 48 h after the PMSG administration and rapidly decreased to the basal level thereafter. Ovarian GnRH mRNA level was slightly decreased at 48 h after the PMSG administration, and then returned to the basal value. GnRH-R mRNA level began to increase at 24 h after the PMSG treatment, decreased below the uninduced basal level at 48 h, and gradually increased thereafter. HCG administration did not alter ovarian GnRH mRNA level, while it blocked the PMSG-induced increase in GnRH mRNA level. Taken together, the present study demonstrates that the expression of GnRH and GnRH-R mRNA are regulated by gonadotropin during follicular development, suggesting possible intragonadal paracrine roles of GnRH and GnRH-R in the mouse ovarian development.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.7
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• pp.1008-1014
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• 2013

This study evaluated the anti-diabetic effects of medicinal plant water extracts on expression of hepatic glucokinase (GCK), pyruvate dehydrogenase (PDH) and acetyl-CoA carboxylase (ACC) mRNA. GCK, PDH and ACC mRNA expression levels were measured by RT-PCR. The medicinal plants used in our study were Cordyceps militaris (CM), Perilla sikokiana (PS), Salvia miltiorrhiza Bunge (SMB), Panax notoginseng (PN) and Angelica utilis Makino (AUM). We found that GCK mRNA expression was increased to about 181% at the 250 ppm of CM water extract. Furthermore, we also found that CM and AUM water extracts stimulated PDH mRNA expression level related to glucose metabolism, however, PS, SMB and PN did not stimulate PDH mRNA expression as expected. Expression of ACC mRNA was also significantly higher in both CM and AUM water extracts. Overall, the results of our study suggest that CM and AUM water extracts stimulate expression of hepatic GCK, PDH and ACC mRNA.

• The Korean Journal of Zoology
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• v.39 no.1
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• pp.115-121
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• 1996

To investigate gestational profiles in gene expression of placental lactogen I fpL4), PL-lI and Pit-1, RNA samples were extracted from the placentas of pregnant day 12 to 20 at 2 day intervals. Northern blots showed changes in gene expression of PL4, - 11 and Pit-i. Sizes of PL-l and -II mRNA were changed and amounts of PL-I, -H and Pit-1 mRNA increased during progress of gestation. To examine the effect of estrogen on the gene expression of PL-I, -Il and Pit-1, pregnant female rats were ovariectomized (OVX) and daily injected with estradiol (OVX + E). OVX markedly lowered the amount of PL4 and 41 mRNA, and shifted niRNA size from 1 kb to i 3 kb in PL-l mRNA and 0.6 kb to i kb in PL-ll mRNA, respectively. OVX had no effect on the mRNA size of Pit-1, but markedly attenuated Pit-1 mRNA level. Estrogen injection reversed the effect of OVX on the size-shift but not on the amount of PL4 and -Il mRNA. Replacement of E partially recovered OVX-induced inhibition of Pit-i mRNA level. Present results suggest that estrogen may play a pivotal role on the gene expression of PL-l and -Il such as alternative RNA splicing and/or polyadenylation, and Pit-1 may be involved in the gene expression of PL-l and 41 by estrogen.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.722-729
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• 2004

Leptin is an adipocyte-secreted hormone and its plasma levels correlate with total body fat mass, however, it also plays a regulatory role in immunity, inflammation, and hematopoiesis. Chemokine is known as a chemoattractant cytokine in inflammatory reaction, but its role in leptin reaction has not been well studied. In this study, the direct effect of leptin on the expression of chemokine mRNAs and lipopolysaccharide (LPS)-induced chemokine KC mRNA in mouse peritoneal macrophages was investigated. Leptin did not induce the expression of lymphotactin, RANTES, eotaxin, MIP-1$\beta$, MIP-1$\alpha$, MIP-2, MCP-1, IP-10, TCA-3, and KC mRNA in mouse peritoneal macrophages, and had no direct effect on the expression of these LPS-induced chemokine mRNAs except KC mRNA. The synergistic effect of leptin on the expression of LPS-induced KC mRNA occurred late in the time course of response to LPS. The increased expressions of Ob-Rb mRNA and leptin receptor protein were detected during the LPS treatment. Leptin produced a substantial increase in the stability of the LPS-induced KC mRNA, and the synergistic effect of leptin on LPS-induced KC mRNA expression was further augmented by cycloheximide (CHX). Pyrrolidine dithiocarbamate (PDTC) did not block the synergistic effect of leptin on LPS-induced KC mRNA expression in mouse peritoneal macrophages. These data suggest that although leptin has no direct effect on the expression of lymphotactin, RANTES, eotaxin, MIP-1$\beta$, MIP-1$\alpha$, MIP-2, MCP-1, IP-10, TCA-3, and KC mRNA in mouse peritoneal macrophages, the synergistic effect of leptin on the expression of LPS-induced KC mRNA has the possibility that LPS might induce the expression of the Ob-Rb receptor or an unknown gene(s) that sensitizes macrophages to the synergistic function of leptin. Therefore, further studies are necessary to examine leptin as a regulatory factor of chemokine production.

• Journal of Environmental Health Sciences
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• v.32 no.6
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• pp.566-570
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• 2006

Exposure to ultraviolet B(UVB) radiation causes skin inflammation such as pigmentation and the induction of cyclooxygenase-2(COX-2) gene expression. In this study, we investigated the effect of natural extracts from Tea, EGb 761 and Korean red ginseng(KRG), on the pigmentation and expression of COX-1 and COX-2 mRNA in UVB-irradiated C57BL/6 mice. Before UVB irradiation, the skin color was significantly showed the lightening effect by topical application of natural compounds (p<.05). In the case of UVB irradiated mice, we observed a decrease in pigmentation by compounds (p<.05). In irradiated skin, COX-1 mRNA expression is not changed following UVB irradiation, but COX-2 gene increases. Also, natural compounds lowered mRNA levels of COX-2. Therefore, these results suggest that COX-2 mRNA increases by UVB irradiation. Also, Tea, EGb 761 and KRG as a topical application may inhibit skin pigmentation and modulate COX-2 mRNA level.

• BMB Reports
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• v.56 no.9
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• pp.514-519
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• 2023

Methyltransferase-like 3 (METTL3), a key component of the m⁶A methyltransferase complex, regulates the splicing, nuclear transport, stability, and translation of its target genes. However, the mechanism underlying the regulation of METTL3 expression by alternative splicing (AS) remains unknown. We analyzed the expression pattern of METTL3 after AS in human tissues and confirmed the expression of an isoform retaining introns 8 and 9 (METTL3-IR). We confirmed the different intracellular localizations of METTL3-IR and METTL3 proteins using immunofluorescence microscopy. Furthermore, the endogenous expression of METTL3-IR at the protein level was different from that at the mRNA level. We found that 3'-UTR generation by intron retention (IR) inhibited the export of METTL3-IR mRNA to the cytoplasm, which in turn suppressed protein expression. To the best of our knowledge, this is the first study to confirm the regulation of METTL3 gene expression by AS, providing evidence that the suppression of METTL3 protein expression by IR is an integral part of the mechanism by which 3'-UTR generation regulates protein expression via inhibition of RNA export to the cytoplasm.

• Tuberculosis and Respiratory Diseases
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• v.41 no.5
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• pp.462-474
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• 1994

Background : In acute lung injury, activated neutrophils play an important role in tissue damage. For neutrophils to participate in lung inflammation, chemotactic factors released from mononuclear phagocytes are needed to bring these cells to the local site of inflammation, with interleukin-8 (IL-8) being one of the most specific and important chemotactic factors for neutrophils. IL-8 also induces the expression of adhesion molecules and activates neutrophils to release various inflammatory mediators. Lipopolysaccharide(LPS) is one of the most important causes of adult respiratory distress syndrome and can cause release of many inflammatory cytokines including IL-8 leading to acute lung injury. But little is known about the regulatory mechanism of LPS-induced IL-8 gene expression in mononuclear phagocytes. Method : Human alveolar macrophages(HAM) and peripleral blood monocytes(PBMC) were isolated from healthy volunteers. Time and dose relationship of LPS-induced IL-8 mRNA expression was observed by Northern blot analysis. To evaluate the regulatory mechanism of LPS-induced IL-8 gene expression, pretreatment of actinomycin D(AD, $5{\mu}g/ml$) and cycloheximide(CHX, $5{\mu}g/ml$) was done and Northern blot analysis for IL-8 mRNA and ELISA for immunoreactive IL-8 protein in culture supernatant were performed. Results : 1) In HAM, dose and time dependent LPS-induced IL-8 mRNA expression was observed with peak mRNA level at 8 hours post-stimulation. 2) In PBMC, dose and time dependent LPS-induced IL-8 mRNA expression was also observed with peak mRNA level at 4 hours post-stimulation. 3) AD decreased expression of LPS-induced IL-8 gene expression at both mRNAand protein levels in both types of cells. 4) CHX decreased expression of LPS-induced IL-8 gene expression at protein level in both cell types but in HAM, superinduction of IL-8 mRNA was observed while decreased expression of IL-8 mRNA was observed in PBMC. Conclusion : Time and dose dependent LPS-induced IL-8 gene expression was observed in mononuclear phagocytes which is at least partly regulated pretranslationally. LPS-induced IL-8 mRNA expression in HAM needs no de novo protein synthesis and may be under the control of a labile repressor protein while de novo protein synthesis may be needed in PBMC.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.685-689
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• 2013

Objective: To study the effects of down-regulation of HDAC6 expression on proliferation, cell cycling and migration of esophageal squamous cell carcinoma (ESCC) cells and related molecular mechanisms. Methods: ESCC cell line EC9706 cells were randomly divided into untreated (with no transfection), control siRNA (transfected with control siRNA) and HDAC6 siRNA (transfected with HDAC6 small interfering RNA) groups. Effects of HDAC6 siRNA interference on expression of HDAC6 mRNA and protein in EC9706 cells were investigated by semi-quantitative RT-PCR, Western blotting and immunocytochemistry methods. Effects of down-regulation of HDAC6 expression on cell proliferation, cell cycle, and cell migration were studied using a CCK-8 kit, flow cytometry and Boyden chambers, respectively. Changes of mRNA and protein expression levels of cell cycle related factor (p21) and cell migration related factor (E-cadherin) were investigated by semi-quantitative RT-PCR and Western blotting methods. Results: After transfection of HDAC6 siRNA, the expression of HDAC6 mRNA and protein in EC9706 cells was significantly downregulated. In the HDAC6 siRNA group, cell proliferation was markedly inhibited, the percentage of cells in G0/G1 phase evidently increased and the percentage of cells in S phase decreased, and the number of migrating cells significantly and obviously decreased. The mRNA and protein expression levels of p21 and E-cadherin in the HDAC6 siRNA group were significantly higher than those in the untreated group and the control siRNA group, respectively. Conclusions: HDAC6 siRNA can effectively downregulate the expression of HDAC6 mRNA and protein in EC9706 cells. Down-regulation of HDAC6 expression can obviously inhibit cell proliferation, arrest cell cycling in the G0/G1 phase and reduce cell migration. The latter two functions may be closely related with the elevation of mRNA and protein expression of p21 and E-cadherin.