[show abstract][hide abstract] ABSTRACT: A novel method is reported for the detection of avian influenza virus subtype H5 using a biosensor based on high spatial resolution imaging ellipsometry (IE). Monoclonal antibodies specific to H5 hemagglutinin protein were immobilized on silicon wafers and used to capture virus particles. Resultant changes on the surface of the wafers were visualized directly in gray-scale on an imaging ellipsometry image. This preliminary study has shown that the assay is rapid and specific for the identification of avian influenza virus subtype H5. Compared with lateral-flow immunoassays, this biosensor not only has better sensitivity, but can also simultaneously perform multiplexed tests. These results suggest that this biosensor might be a valuable diagnostic tool for avian influenza virus detection.
[show abstract][hide abstract] ABSTRACT: To study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.
An eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.
A stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.
Exogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 03/2006; 28(2):103-6.
[show abstract][hide abstract] ABSTRACT: Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2004; 20(3):332-6.
[show abstract][hide abstract] ABSTRACT: BRCA1 is one of the most important breast cancer susceptibility genes. It plays key roles in DNA damage repair, cell cycle checkpoint regulation, gene transcription chromatin stability, and cell proliferation. In this paper, advance in basic researches on BRCA1 is reviewed, and the role of BRCA1 in cancer development and progression is discussed, that may facilitate potential clinical application of BRCA1.
[show abstract][hide abstract] ABSTRACT: Estrogen receptor (ERalpha) plays an important role in the development and progression of breast cancer. Several recent studies have demonstrated that expression of human X-box binding protein 1 (XBP-1) is associated with ERalpha status in breast tumors and overexpressed in a subset of breast tumors. XBP-1 has two splicing variants, which were designated as XBP-1S and XBP-1U, respectively. However, little is known about the expression pattern of XBP-1S and XBP-1U in breast cancer cells and about their roles in ERalpha signaling. In this study, the expression of two splicing forms of XBP-1 was detected in breast cancer cell lines with RT-PCR. Estrogen response element (ERE) -containing luciferase reporter assay was used to determine the effects of XBP-1S and XBP-1U on the transcription activity of ERalpha in MDA-MB-435 breast cancer cells. The result showed that both XBP-1S and XBP-1U enhanced the transcription activity of ERalpha in a hormone-independent and dose-dependent manner and the activity of of XBP-1S is higher than that of XBP-1U. Enhancement of ERE-containing luciferase reporter gene expression by XBP-1S and XBP-1U was dependent on ERalpha. These data suggest that XBP-1S and XBP-1U may play important roles in breast cancer growth and progression through ERalpha signaling.
[show abstract][hide abstract] ABSTRACT: BRCA1 (breast cancer susceptibility gene-1) plays important roles in DNA damage repair, cell checkpoint regulation, gene transcription, chromosome stability, and apoptosis. At the C-terminus of BRCA1 is the activation domain with a number of acidic amino acid residues that includes two tandem repeats of BRCT(BRCT1 and BRCT2). In this study, to identify proteins that interact with the BRCT2 domain of BRCA1, the standard yeast two-hybird screen was performed. FHL2 was isolated from a human ovary library, with the BRCT2 domain of BRCA1 as bait. Furthermore, the specific interaction of FHL2 with the BRCT2 domain of BRCA1, but not with the BRCT1 domain of BRCA1 and the BRCT domain of Rap1, was verified by yeast mating. To confirm the interaction between BRCA1 and FHL2 in vitro, the GST pull-down assay was performed, the coding sequences of BRCT1 and BRCT2 domains were fused in-frame with the coding region of GST in the pGEX-2T vector, generating the pGST-BRCT1 and pGST-BRCT2 recombinant plasmids the fusion proteins GST-BRCT1 and GST-BRCT2 were expressed in E. coli DH5 alpha. The purified fusion proteins were obtained by GST-Sepharose 4B affinity chromatography. The purified fusion proteins were incubated with in vitro translated 35S-methinine-labeled FHL2. Consistent with the two-hybird results, FHL2 could specifically bind to the BRCT2 domain, but not BRCT1 in vitro. To further assess the binding specificity of FHL2 to the BRCT2 domain of BRCA1 in vivo, pFLAG-FHL2 and pHABRCT1/pHA-BRCT2 recombinant plasmids were cotransfected into 293T cells. Then the coimmunoprecipitation assay were performed. The results also showed that FHL2 specifically interacted with the BRCT2 domain in vivo. Furthermore, the coimmunoprecipitation assay demonstrated that FHL2 could interact with endogenous BRCA1 in vivo. These findings lay solid foundations for study on the function of BRCA1 and FHL2 in cancer development and progression.
[show abstract][hide abstract] ABSTRACT: The estrogen receptor (ERalpha) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that human X-box binding protein 1 (XBP-1) mRNA expression is associated with ERalpha status in breast tumors. More recently, two forms of XBP-1 were identified due to their unique splicing. The two splicing variants of XBP-1 were designated XBP-1S and XBP-1U, respectively. In this study, the coding sequences of XBP-1S and XBP-1U were cloned respectively into the expression vector pcDNA3 harboring FLAG epitope, generating the recombinant plasmids pcDNA3-FLAG-XBP-1S and pcDNA3-FLAG-XBP-1U. Western blot analysis showed that both XBP-1S and XBP-1U were expressed in mammalian cells. To determine the effects of XBP-1S and XBP-1U on the transcriptional activity of ERalpha, MDA-MB-231 breast cancer cells were cotransfected with the expression vectors for ERalpha and either pcDNA3-FLAG-XBP-1S or pcDNA3-FLAG-XBP-1U. The results indicated that XBP-1S and XBP-1U enhanced ERalpha-mediated transcriptional activities in a hormone-independent manner. GST pull-down assay showed that both XBP-1S and XBP-1U interacted with ERalpha. These data suggest that XBP-1S and XBP-1U may play an important role in breast cancer growth and progression through ERalpha signaling.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 10/2003; 35(9):829-33.
[show abstract][hide abstract] ABSTRACT: To construct an ERbeta expression vector and study its expression and function in different cancer cells.
Standard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements.
The recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3.
ERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 08/2003; 25(4):340-3.
[show abstract][hide abstract] ABSTRACT: FHL2, a member of LIM-only protein family, plays an important role in transcription regulation, apoptosis, cancer development and progression. In this study, a mammalian transcription activation system was constructed by using DNA binding domain(DBD) of GAL4 and luciferase reporter gene with DBD binding sequence, and used for mapping of FHL2 transcription activation domain. First, the coding sequence of GAL4-DBD was inserted into expression vector pcDNA3, generating the pDBD recombinant plasmid, then the wild-type FHL2 and its mutants were fused in-frame with GAL4-DBD, resulting in expression vectors for wild-type FHL2 and its mutants. All of the recombinant plasmids were transfected into 293T cells. Western blot assay showed that all of the fusion proteins were expressed. The analysis of FHL2 transcription activation properties by using the GAL4-luciferase reporter gene indicated that wild-type FHL2 had activation activity in both 293T and MCF-7 cells. The deletion of the half LIM domain at the N-terminus severely impaired the capacity of FHL2 to stimulate transcription. The mutant lacking the LIM domain at the C-terminus was totally inactive, while the deletion of two LIM domains at the C-terminus partially recovered its ability to stimulate transcription. The deletion of the second LIM domain at C-terminus did not alter the activation capacity of FHL2. These results suggest that the last LIM domain at the C-terminus of FHL2 is critical for its transcription activation function, the second LIM domain at the C-terminus may be a negative regulation region, but this negative regulation depends on the last LIM domain. Mapping of transcription activation domain of FHL2 lays solid basis for further study on various FHL2 functions.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 08/2003; 35(7):643-8.