• BMB Reports
• /
• v.45 no.2
• /
• pp.126-131
• /
• 2012

We have previously identified a DNA silent region located downstream of the 3'-end of the ${\beta}^{major}$ globin gene (designated B1-559) that contains a B1 retrotransposon, consensus binding sites for erythroid specific transcription factors and shares the capacity to act as promoter in hematopoietic cells interacting with ${\beta}$-globin gene LCR sequences in vitro. In this study, we have cloned four new non-polyA RNA transcripts being detected upon blockade of murine erythroleukemia (MEL) cell differentiation to erythroid maturation by methylation inhibitors and demonstrated that two of them share high structural homology with sequences of B1 element found within the B1-559 region. Although it is not clear yet whether and how these RNAs interfere with induction of erythroid maturation, these data provide evidence for the first time showing that methylation inhibitors can activate silent repetitive DNA sequences in MEL cells and may have implications in cancer chemotherapy using demethylating drugs as antineoplastic agents.

• Journal of Life Science
• /
• v.12 no.2
• /
• pp.208-212
• /
• 2002

Human $\varepsilon$-globin DNA fragment was used to determine the thermal stabilities of base pairs at d(CXG) and d(GXC) by Temperature Gradient Gel Electrophoresis(TGGE). The base pair stability depends on the hydrogen bonding interaction and base stacking interaction of neighbor base sequence. The orders of base pair stabilities were T.AG.A = A.G>C.T>T.C>C.A>A.C for d(GXC).d(GYC).

• International Journal of Oral Biology
• /
• v.35 no.2
• /
• pp.61-67
• /
• 2010

Selenoprotein S (SelS) is widely expressed in diverse tissues where it localizes in the plasma membrane and endoplasmic reticulum. We studied the potential function of SelS in erythrocyte differentiation using K562 cells stably over-expressing SelS wild-type (WT) or one of two SelS point mutants, $U_{188}S$ or $U_{188}C$. We found that in the K562 cells treated with $1\;{\mu}M$ Ara-C, SelS gradually declined over five days of treatment. On day 4, intracellular ROS levels were higher in cells expressing SelS-WT than in those expressing a SelS mutant. Moreover, the cell cycle patterns in cells expressing SelS-WT or $U_{188}C$ were similar to the controls. The expression and activation of SIRT1 were also reduced during K562 differentiation. Cells expressing SelS-WT showed elevated SIRT1 expression and activation (phosphorylation), as well as higher levels of FoxO3a expression. SIRT1 activation was diminished slightly in cells expressing SelS-WT after treatment with the ROS scavenger NAC (12 mM), but not in those expressing a SelS mutant. After four days of Ara-C treatment, SelS-WT-expressing cells showed elevated transcription of $\beta$-globin, $\gamma$-globin, $\varepsilon$-globin, GATA-1 and zfpm-1, whereas cells expressing a SelS mutant did not. These results suggest that the suppression of SelS acts as a trigger for proerythrocyte differentiation via the ROS-mediated downregulation of SIRT1.

• Proceedings of the Zoological Society Korea Conference
• /
• 1996.10a
• /
• pp.219.1-219
• /
• 1996

No Abstract, See Full Text

• Proceedings of the Zoological Society Korea Conference
• /
• 1996.10a
• /
• pp.218.1-218
• /
• 1996

No Abstract, See Full Text

• The Korean Journal of Zoology
• /
• v.39 no.3
• /
• pp.239-247
• /
• 1996

An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.

• Journal of Applied Biological Chemistry
• /
• v.53 no.4
• /
• pp.189-194
• /
• 2010

We confirmed the hypo-osmotic shock strengths and duration, different type of vectors, and subcelluar localization to identify the optimum analysis condition of plant aquaporin activity in Xenopus ooctye using Arabidopsis thaliana AtPIP2-1 gene. Six minutes and 1/5ND buffer hypoosmotic shock treatment was the best condition to show the maximum swelling of Xenopus oocytes where AtPIP2-1 was expressed using pcDNA3.1 vector. AtPIP2-1 protein was expressed more efficiently in pGEMHE vector which has 5' and 3' UTR (untranslation region) of Xenopus ${\beta}$-GLOBIN gene in multiple cloning site than in pcDNA3.1 vector. Also green fluorescence of GFP fused to AtPIP2-1 was detected onto oocyte plasmamembrane where is the proper subcellular localization target of AtPIP2-1.

• Reproductive and Developmental Biology
• /
• v.31 no.4
• /
• pp.241-248
• /
• 2007

Epigenetic modification dependent DNA methyltransferases (DNMTs) play an important role in tissue- and stage-specific gene regulation and normal mammalian development. In this study, we show that DNMTs are expressed at different levels during hematopoietic stem cell (HSC) differentiation to proerythrocytes. DNMT1, DNMT3A, and DNMT3B were highly expressed at day 7 after differentiation. We used specific siRNA as a tool to probe the relationship between the expression of DNMTs and erythropoietic differentiation. When introduced siRNA of DMNT1 and DMNT3b in human $CD34^+$ cells, these more differentiated into erythrocytes. This was confirmed by glycophorin A (GPA) positive cell analysis and globin gene expression. $GPA^+$ cells increased up to $20{\sim}30%$, and ${\gamma}$- and ${\epsilon}$-globin genes increased in siRNA transfected cells. Therefore, our data suggest that suppression of DNA methylation can affect positively differentiation of HSC and may contribute to expression of erythrocyte lineage genes including GPA and globins.

• Journal of Environmental Health Sciences
• /
• v.32 no.2 s.89
• /
• pp.164-170
• /
• 2006

Ethylene oxide is a genotoxic carcinogen with widespread uses as industrial chemical intermediate and gaseous sterilant. 2-hydroxyethylated N-terminal valine in Hb is a good biomarker for biological monitoring of ethylene oxide exposure, because of its stability. For measuring the hemoglobin adduct formed by exposure of ethylene oxide, we studied the determination of (N-2-hydroxy-ethyl)valine(HEV) in hemoglobin adduct by using GC/MS. Firstly we synthesized HEV with 2-amino-ethanol and bromoisovaleric acid(BIVA) and confirmed it with GC/MS-FID. Its fragmentations were m/z 116(base ion, M+-45) and m/z 130(M+-31). For measuring HEV with higher sensitivity, we use derivatives which were PFPITH(pentafluorophenylisothiocianate) and TBDMS (tributyldimethylsilylation) by using Edman procedure. Its fragmentation were m/z 425(M+-57), m/z 383(M+-99) and m/z 172(M+-310) by using GC/MS. We did biological monitoring for mice inhalation exposure with 400 ppm ethylene oxide. The concentrations of hemoglobin adduct were $168{\pm}3.8\;and\;512{\pm}04$(nmol g-1 globin) at 0.5 hr/day 400 ppm ethylene oxide inhalation exposure group, and $631{\pm}17\;and\;2265{\pm}9.4$(nmol g-1 globin) at 1.0 hr/day 400 ppm ethylene oxide inhalation exposure for 1 and 4 weeks, respectively. We confirmed that (N-2-hydroxy-ethyl)valine(HEV) of hemoglobin was a good biomarker for biomonitoring of ethylene oxide exposure, and can measured with derivatives such as PFPITH(pentafluorophenylisothiocianate) and TBDMS(tributyldimethylsilylation) by using GC/MS.

• The Korean Journal of Zoology
• /
• v.27 no.4
• /
• pp.199-212
• /
• 1984

A cytoplasmic endoprotease, named protease Ci, has been partially purified by classical chromatographic procedures. This enzyme degrades insulin, glucagon and bovine growth hormone to trichloroacetic acid-soluble materials, but shows little or no hydrolysis of bovine serum albumin, casein or globin. It has a molecular weight of about 120,000 as determined by gel filtration on Sephadex G200, and it appears to be consisted of two identical subunits having molecular weight of 54,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Protease Ci has an optimum pH of 7.5, and has an isoelectric point of 5.5. This enzyme is a metalloprotease, since it is inhibited by o-phenanthroline and can be activated by the addition of divalent metal cations, such as $Mn^++$ and $Co^++$. Protease ci is inhibited by p-hydroxymercuribenzoic acid, but not by either of leupeptin or Ep475 which are specific inhibitors of sulfhydryl protease. It is distinct from protease Pi, a perplasmic insulin degrading enzyme, since protease Ci is localized to the cytoplasm. The physiological function of protease Ci is presently unknown.