- Transcription Factor Profile by Degenerate RT-PCR/SSCP: Application in 3T3-L1 Adipocyte Treated with TNF-alpha.
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Yoo Lee Kim, Sang Hwa Lee, Young Kil Choi, Seo Yoon Chang, Yun Soo Kim, Soo Kyung Kim, Seok Won Park, Won Kun Park, Yong Wook Cho, Sang Jong Lee
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Korean Diabetes J. 2007;31(5):410-420. Published online September 1, 2007
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DOI: https://doi.org/10.4093/jkda.2007.31.5.410
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 Abstract
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- BACKGROUND
Several high-throughput gene analysis techniques - differential display PCR, suppression subtraction hybridization (SSH), serial analysis of gene expression (SAGE), and DNA microarray - have permitted transcriptome profiling to understand the molecular pathogenesis of multifactorial diseases. But these techniques are of no great utility regarding feasibility, reproducibility, cost, and the amount of material required for analysis. To establish more practical method for transcription factor transcriptome profiling, we combined degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and single strand conformational polymorphism (SSCP) technique. METHODS: We categorized 417 human/mouse transcription factor mRNA into 92 small groups according to homology with ClustalW method and established 92 degenerate RT-PCR including common motives of the 92 small groups with the software program of CODEHOP, Primer Premier, Amplify 1.2. Further analysis on the amplified PCR products was performed by SSCP. This system was applied for the evaluation of changes on transcription factor transcriptome of differentiated 3T3-L1 adipocyte treated with TNF-alpha. RESULTS: 82 groups and 52 groups showed amplification of PCR before and after TNF-alpha treatment respectively and 24 groups showed significant amplification difference after TNF-alpha treatment. After TNF-alpha treatment for 48 hours, mRNA expressions of group 7, 30, and 33 which include adipocyte related transcription factors such as CEBP-alpha, RXR-alpha, PPAR-gamma were downregulated and mRNA expression of group 8 including preadipocyte abundant CEBP-beta was upregulated. These results are largely concordant with the results analyzed by oligonucleotide microarray. Randomly selected single PCR bands of group 28 and 75 on agarose electrophoresis displayed additional multiple bands by SSCP and necessitated addition of this technique to degenerate RT-PCR for further analysis. CONCLUSION: It could be suggested that degenerate RT-PCR/SSCP is practical method and could be used as a screening test for transcriptome profiling of various disease states with further validation study.
- Clinical Courses of Two Women with Gestational Diabetes Mellitus Who are GAD Antibody Positive.
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Sung Hoon Yu, Min Jun Song, Sung Hoon Kim, Chang Hoon Yim, Ki Ok Han, Won Kun Park, Hyun Koo Yoon, Ho Yeon Chung
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Korean Diabetes J. 2006;30(5):398-402. Published online September 1, 2006
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DOI: https://doi.org/10.4093/jkda.2006.30.5.398
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Abstract
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- Gestational diabetes mellitus (GDM) is defined as glucose intolerance of various degrees with onset or first recognition during pregnancy. Women with GDM are at high risk of developing type 2 diabetes later in life, but the risk of developing type 1 diabetes is also increased. Positivity for glutamic acid decarboxylase (GAD) antibodies during pregnancy confers a high risk for subsequent progression to type 1 diabetes. Here, we reported the two cases with GDM who were GAD antibody positive and progressed to type 1 diabetes with different time-courses. One woman with GDM progressed rapidly to classical type 1 diabetes while the other became slowly progressive IDDM (SPIDDM) [or latent autoimmune diabetes in adults (LADA)].
- Alteration of Hypertonic Responses in Hyperglycemic Human Retinal Pigment Epithelial and Placental Cells.
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Won Kun Park
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Korean Diabetes J. 2004;28(3):164-176. Published online June 1, 2004
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Abstract
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- BACKGROUND
In mammals, cellular protection from hypertonic damage is achieved by increasing the expression of genes, such as aldose reductase(AR) and Na+/myo-inositol transporter(SMIT). Recent studies have revealed that tonicityresponsive enhancer binding protein(TonEBP) regulates the increased transcription of these genes. Increased AR gene expression leads to hyperglycemiainduced cellular impairment, and the degree of basal aldose reductase gene expression influences the development of diabetic complication. The alteration of cellular protection genes, such as AR, SMIT, HSP(Heat Shock Protein) 70 and TonEBP, from hypertonic stress in the presence of sustained hyperglycemia was examined in human retinal pigment epithelial(hRPE) and placental cells. METHODS: After the cultures became confluent, the hRPE cells were exposed to 25mM glucose, 100mM NaCl or both for 1, 2 and 3 days. The expressions of AR, SMIT and HSP70 were determined by northern blot analysis. Decreased inductions of the hypertonicity in AR and SMIT mRNA were first evident after exposure to 25mM glucose after 1 day, achieving near maximal level after 3 days; however, no significant alteration in the HSP70 mRNA was noted at any time. For these reasons, the alteration of cellular protection genes, such as AR, SMIT and TonEBP, in hRPE and placental cells were examined after 3-days exposure to the various experimental conditions. The cells were incubated in serum-free media, containing 5.5 or 25mM glucose, 100mM NaCl(in hRPE cells) or 75mM NaCl(in placental cells) and 25mM glucose+100mM NaCl(in hRPE cells) or 75mM NaCl(in placental cells), with or without 20muM tolrestat for 72hrs, at which time the expressions of AR, SMIT and TonEBP were determined. To examine the role of cellular ionic strength in hyperglycemic hRPE cells, excess(5mM) betaine was added to the medium to accelerate the accumulation of the compatible osmolyte, which should lead to a strength reduction. RESULTS: In the hRPE and placental cells, incubated in media with 25mM glucose+100mM(in hRPE cells) or 75mM(in placental cells) NaCl, the AR and SMIT mRNA levels were decreased compared to those cells incubated in media with 100mM(in hRPE cells) or 75mM(in placental cells) NaCl alone. Significant prevention of these decreased AR and SMIT mRNA levels were also observed in those cells incubated with tolrestat. The addition of betaine reduced the abundance of AR and SMIT mRNA in hypertonic stress, similar to in hypertonic and hyperglycemic cells. The inhibition of aldose reductase due to tolrestat in hyperglycemic and hypertonic media was complete in the media not containing betaine. CONCLUSION: Under conditions where sorbitol rapidly increases in hypertonic and hyperglycemic medium should be an important new system for exploring the mechanism of cellular impairment to the effects of hyperglycemia. The expressions of AR and SMIT genes that may influence the development of diabetic complication were down-regulated by the intracellular accumulation of sorbitol in sustained hyperglycemia, and the reliable effect of AR inhibitors on the biological improvements were verified in hyperglycemic cells.
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