Minhyung Lee

Apoptosis induced by progesterone in human ovarian cancer cell line SNU‐840

Progesterone has been used as an ingredient of anticancer drug for patients with ovarian carcinoma. However, the mechanism of anticancer effects by progesterone has not been understood. In this study, the effects of progesterone on ovarian cancer cells, SNU‐840, were investigated. After the incubation with progesterone, the viability of the cells was evaluated by MTT assay. As a result, 45% of the cells were viable after 48 h of incubation with 100 μM progesterone. In addition, [3H]thymidine incorporation assay showed that the proliferation of the cells was completely inhibited by progesterone after 48 h of incubation at 100 μM concentration. Colorimetric TUNEL assay revealed the fragmentation of the chromosomal DNA, suggesting that the process of the cell death was apoptosis. The level of the p53 mRNA was determined by northern blotting assay, since many apoptosis processes are mediated by up‐regulation of the p53 expression. The level of the p53 mRNA reached its maximum at 12 h and decreased after 24 h of incubation with progesterone. In conclusion, progesterone inhibits the proliferation and elicites apoptosis of SNU‐840 cells. Also, it up‐regulates the p53 mRNA transiently. J. Cell. Biochem. 82:445–451, 2001.

Transcription of the Rat p53 Gene Is Mediated by Factor Binding to Two Recognition Motifs of NF1-Like Protein

In this study we analyzed the ratp53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis. As a result we identified two protein binding elements (element 1: -296 to -312, element 2: -195 to -219) with sequence homology to each other. The two identified elements bind to the same kind of protein. To identify the protein binding to these elements, competition assays were carried out with double stranded oligonucleotides containing NF1, YY1, and CRE consensus motifs. Only the NF1 consensus motif competed with element 1 and 2. Element 2 is conserved between the rat, human, and mouse p53 promoters, and has an NF1 consensus motif. However, the sequences of element 1 are comparatively variable between the species. Only the element 1 region of the rat p53 promoter has partial homology to the NF1 consensus motif. This suggests that the element 1 is specific for the rat p53 gene. The molecular mass of the binding protein, determined by Southwestern blotting analysis, was 40 kDa, which is different from that of NF1. In EMSA with an anti-NF1 antibody, DNA-protein complexes were neither supershifted nor decreased. The 40 kDa protein was also detected in rat spleen and lung, but not in kidney. The binding protein was purified by sequence-specific DNA affinity chromatography and it was confirmed that the purified protein binds to the two regions. It was also proved that the identified two elements are required for basal level transcription of the rat p53 gene by in vitro transcription assay.

Biochemical characterization of a nuclear factor that binds to NF1-like elements in the rat p53 promoter.

We previously reported that two nuclear factor 1‐like elements mediated the transcription of the rat p53 gene. A 40‐kDa protein was shown to bind to these elements, which was different from common NF1 family proteins. In this study, the biochemical properties of the 40‐kDa binding protein were investigated. The metal ion dependency of the protein was examined with various chelators; the protein was proved to require Mg2+ for maximum DNA‐binding activity. The binding protein was highly resistant to ionic strength and denaturant. The protein‐DNA complex was reduced at high NaCl concentration, but residual DNA‐binding activity remained. Even 2 M urea did not completely eliminate the formation of protein‐DNA complex. DNA‐binding activity of the protein was also stable at high temperature. Treatment of the protein‐DNA complex with increasing concentrations of proteinase K or trypsin demonstrated the existence of a protease‐resistant DNA‐bound core. These biochemical properties provide new insight into the 40‐kDa NF1‐like nuclear factor. J. Cell. Biochem. 78:1–7, 2000.

Characterization of a nuclear factor that binds to AP1-like element in the rat p53 promoter during liver regeneration

The transcription level of the rat p53 gene increases at 5–12 h in the regenerating liver after partial hepatectomy. It was previously reported that an activator protein 1 (AP1)‐like element (‐264–‐284) mediated the induced transcription of the rat p53 gene during liver regeneration. In this study, we characterize the protein binding to the AP1‐like element by various methods. Oligonucleotide competition assays showed that the binding protein did not require AP1 consensus sequence. Therefore, the binding protein is not an AP1 family protein. Zn2+ was required for maximum DNA‐binding activity of the protein, suggesting that the binding protein contains zinc fingers. The binding protein was highly resistant to denaturant. Even 1.8 M urea did not eliminate the protein–DNA complexes. In addition, the binding protein was stable up to 55°C. The protein–DNA complexes were abolished in the presence of 0.6 M NaCl and higher. Protease clipping assay showed that the protein had a protease‐resistant core DNA binding domain. These results provided new insights into the structure of the protein that binds to the AP1‐like element of the p53 promoter during liver regeneration. J. Cell. Biochem. 80:124–132, 2000.

An engineered lox sequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision

In our previous report, one 34-bp sequence from a long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) clone, loxLTR-1, was proposed as a target site for site-specific excision by modified Cre recombinase. To support this suggestion, an engineered lox sequence, designated loxIL1, was made. This variant lox has the corresponding sequence of loxLTR-1 at the spacer region and the last two bases of inverted repeat sequence. Through in vitro recombination assay, loxIL1 also allowed the wild-type Cre to specifically recombine the sequence. An in vitro DNA binding experiment with mutants CreK244R and CreK244L revealed that lysine 244 of Cre plays an important role in interaction with the engineered lox. This result suggests that loxLTR-1 would be a candidate for antiviral strategy using site-specific recombinase.

Characterization of nuclear factors binding to AT‐rich element in the rat p53 promoter

In this study, we identified AT‐rich element located at positions −504 to −516 in the rat p53 promoter by DNase I foot printing assay. This region was previously identified as a positive regulatory element in the murine p53 promoter and designated as PBF1 (p53 binding factor 1) binding site. However, the proteins binding to this AT‐rich element have not been identified yet. Therefore, we characterized the binding protein by various biochemical methods. First, we confirmed that by the oligonucleotide competition assay, nuclear factors bound to the AT‐rich element in a sequence‐specific manner. Two binding proteins were identified in southwestern blotting analysis and the molecular masses of the proteins were 60 and 40 kDa, respectively. The proteins were stable to denaturants or ionic strength. Treat‐ment of chelators showed that the binding proteins did not require divalent cation for DNA‐binding activity. In addition, the binding proteins were labile to protease treatment. This study showed that 60 and 40 kDa proteins bound to AT‐rich element and the physico‐chemical properties provided new insights into the binding proteins. J. Cell. Biochem. 80:580–588, 2001.

In vitro transcription assay with the purified 40kDa NF1-like protein binding to the rat p53 promoter.

The rat p53 promoter has several potential transcription factor‐recognition motifs. They inculde NF1‐like, bHLH family, and AP1‐like proteins binding sites. The binding protein to NF1‐like motif was previously identified. The protein has about 40kDa of molecular mass, which is smaller than that of NF1. Anti‐NF1 polyclonal antibody does not recognize the protein. In this study, we isolated the 40kDa protein by sequence‐specific DNA affinity chromatography. The isloated protein was assayed by DNase I footprinting analysis. To determine the transactivation effect of the protein, in vitro transcription with the purified 40kDa protein was carried out. After the addition of the purified 40kDa protein into the transcription reaction mixture, the transcription level of the p53 promoter was increased. This suggests that the 40 kDa NF1‐like protein is a transcription activator for the rat p53 gene.

Transcription of the rat p53 gene is induced by a 39kDa protein binding to the p53 promoter region during the liver regeneration

The adult rat liver is normally in a state of growth arrest. However, cell loss such as partial hepatectomy can induce the proliferation of the hepatocytes. Early after partial hepatectomy, the concentration of p53 mRNA increases during the prereplicative phase. In this study, we identified the cis‐regulatory element involved in the induced transcription of the rat p53 gene by DNase I footprinting assay. This element had a partial homolgy to the AP 1 recognition motif, but the competition study with AP1 oligonucleotide showed that this element was not the AP1 recognition motif. The molecular weight of the binding protein to this motif was determined as 39kDa by southwestern blotting analysis. In vitro transcription assay with the competitor containing the binding motif showed that the 39kDa protein binding to the element was requried for the induced transcription of the rat p53 gene during the liver regeneration.

Characteristics of tinnitus found in anemia patients and analysis of population-based survey

OBJECTIVE This study analyzed the characteristics of tinnitus identified in anemia patients with cohort- and population-based studies in a single institute and suggests a management algorithm. METHODS Fifty patients who were treated for anemia and referred for tinnitus treatment were included in a single institute retrospective study. Characteristics of tinnitus were investigated in a correlation analysis with demographic and audiologic parameters. For the population-based study, data from the Korea National Health and Nutrition Examination Survey collected between 2010 and 2011 were analyzed. The study population consisted of 11,402 individuals aged 20-97 years with complete tinnitus-related data. The prevalence of tinnitus in anemia patients was investigated using the questionnaire, and associations between tinnitus and blood/urine parameters were evaluated by binary logistic regression analysis. RESULTS In a single-institute study, patients with non-pulsatile tinnitus were significantly older and their initial hemoglobin was higher than those with pulsatile tinnitus (p=0.001, 0.008, respectively). In pulsatile tinnitus, age and difference between initial and post-treatment hemoglobin were significantly associated with a subjective improvement in tinnitus (p=0.002, 0.016, respectively). There were no significant audiologic or hematologic parameters associated with the improvement of non-pulsatile tinnitus. In the population-based study, there was no significant correlation between anemia and tinnitus (p=0.064). In a multivariate analysis, age was the only parameter associated with tinnitus in participants with anemia. CONCLUSION The therapeutic strategy and prognosis of tinnitus in anemia patients differ according to the characteristics of tinnitus and the severity of anemia.

Design of a windbreak fence to reduce fugitive dust in open areas

The wind characteristics of the Saemangeum reclaimed land are quite different from those of inland because of the prevalence of strong wind environments near the seashore. Thus, the probability of fugitive dust dispersion would be higher under these wind environmental conditions, and civil complaints regarding fugitive dust have increased in the residential area. Therefore, we must establish countermeasures to reduce fugitive dust. In this study, the dispersion behavior of fugitive dust at the Saemangeum reclaimed land was simulated by using a two-dimensional CFD simulation. A CFD model was simulated by considering the wind velocity and the design properties of the windbreak fences, such as their height, installation interval and number of layer. The percent of upstream velocity (PUV) is applied in this study to quantitatively evaluate the reduction in the wind velocity by a windbreak fence. The behavior of saltating dust was analyzed by comparing the simulation results with the threshold wind velocity, which is the wind velocity at which the soil particulates come off the surface and erosion starts. The behavior of suspended dust was analyzed by using the reduction rate of dust concentration according to the installation conditions of the windbreak fence. A strategy for installing windbreak fences was suggested according to these results to reduce fugitive dust in the Saemangeum reclaimed land.