Browsing by Author "Lee, I-Liang"

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alpha B-Crystallin in clear cell renal cell carcinoma: Tumor progression and prognostic significance
(2013-10-01)
Ho, Pei-Yin
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Chueh, Shih-Chieh
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Chiou, Shyh-Horng
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Wang, Shuo-Meng
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Lin, Wei-Chou
Objectives: AlphaB-crystallin (alpha B-crystallin), a small heat shock protein, has been reported to be involved in the growth, antiapoptosis, migration, and chemoresistance of human malignancies. Materials and methods: alpha B-crystallin expression in normal renal and clear cell renal cell carcinoma (ccRCC) tissues was examined with two-dimensional (2D) gel electrophoresis assays. Immunohistochemistry was conducted to determine the presence of alpha B-crystallin-positive tumor cells and staining intensity in 50 cases of ccRCC tissue samples. The association of alpha B-crystallin protein expression, clinicopathogic parameters and prognosis of ccRCC patients was also analyzed with Student's t-test and Kaplan-Meier analysis. Moreover, Western blot assays were performed to detect the protein expression of alpha B-crystallin in normal and tumor tissues and the alteration of cell cycle regulators in alpha B-crystallin-overexpressing cells. MTT (3[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide), BrdU, and transwell assays were performed to demonstrate the effects of aB-crystallin overexpression on cell growth, DNA synthesis and cell migration of ccRCC cells, respectively. Results: The results showed the up-regulation of alpha B-crystallin expression in ccRCC tissues. Overall survival of ccRCC patients was significantly correlated with alpha B-crystallin expression in tumor tissues. We found that aB-crystallin overexpression increased the expression of cyclin A and the incorporation of BrdU, which may be related to the enhancement of cell growth. Transwell analyses demonstrated that presence of alpha B-crystallin overexpression enhanced cell migration in ccRCC cells. Furthermore, rapamycin-resistance of tumor cells was induced when alpha B-crystallin was overexpressed. Conclusions: Our experimental findings highlight the importance of alpha B-crystallin in the tumor growth, migration, and target therapy-resistance of ccRCC cells. (C) 2013 Elsevier Inc. All rights reserved.
Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts
(2011-09-01)
Kuan, Tang-Ching
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Yang, Tzu-Hui
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Wen, Cheng-Hao
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Chen, Mu-Yuan
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Lee, I-Liang
Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1-7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position -2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the -516/-481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA-protein complex with double stranded oligonucleotides of the -516/-481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II-Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-beta 1 and TNF-alpha. Our results suggest that the nucleotide sequences -516/-481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation. (C) 2011 Elsevier Inc. All rights reserved.
Prokaryotic expression, refolding, and purification of functional human vascular endothelial growth factor isoform 165: Purification procedures and refolding conditions revisited
(2011-03-01)
Lee, I-Liang
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Li, Pei-Shan
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Yu, Wei-Lin
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Shen, Hsin-Hsin
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生物科技學系
Human vascular endothelial growth factor isoform 165 (VEGF165) is the first known member belonging to the VEGF protein family that plays a critical role in new blood vessel formation in vivo. This study presents a new protocol with optimized conditions for rapidly producing untagged recombinant and biological active human VEGF165 (rhVEGF165) using Escherichia coli cells. Protein was isolated from inclusion bodies, purified by gel filtration and ion exchange chromatography, and subjected to protein refolding and renaturation. The biological activity of rhVEGF165 is comparable with VEFG from eukaryotic source according to human umbilical vein endothelial cells (HUVEC) proliferation assay. Therefore, the present procedures provide a fast and easy way to produce this therapeutic protein. (C) 2010 Published by Elsevier Inc.
Synthesis and interfacing of biocompatible iron oxide nanoparticles through the ferroxidase activity of Helicobacter Pylori ferritin
(2012-12-01)
Lee, I-Liang
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Li, Pei-Shan
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Yu, Wei-Lin
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Shen, Hsin-Hsin
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生物科技學系
Ferritin is an iron storage protein that is often used to coat metallic nanoparticles, such as iron oxide nanoparticles (IONPs). However, the synthesis and biocompatibility of ferritin-coated IONPs remain unclear. Therefore, this study reports the synthesis of a ferritin gene cloned and expressed from Helicobacter pylori (HPFn). The ferroxidase activity of the synthase HPFn was used for the de novo synthesis of HPFn-coated IONPs under mild conditions. Gel filtration chromatography and transmission electron microscopy analyses demonstrated that the core-shell structure of both the 5.0 nm IONP nanocore and the 12.4 nm HPFn shell were correctly assembled. The cellular uptake of mouse macrophage cells (RAW 264.7 cells) has shown that only a few HPFn-coated IONPs (3%) were taken up after 24 h of incubation. This study compares the biocompatibility of HPFn-coated IONPs, superparamagnetic iron oxide nanoparticles (SPIOs) and ferric salt (ferric ammonium citrate) in respect to cell growth inhibition, reactive oxygen species generation and pro-inflammatory cytokine TNF-alpha release. Assessment results showed that the responses elicited by HPFn-coated IONPs were similar to those elicited by SPIO treatment but milder than those elicited by ferric salt treatment. This accounts for the notion that ferritin-coated IONPs are biocompatible iron agents. These findings show that the ferroxidase activity of ferritin can be used to synthesize biocompatible IONPs. The favorable properties of HPFn-coated IONPs suggest that they can be used as a non-macrophage contrast agent through further surface conjugation.