DSpace community: 口腔生物科學研究所

Intronic Enhancers Control Expression of Zebrafish Sonic Hedgehog in Floor Plate and Notochord

title: Intronic Enhancers Control Expression of Zebrafish Sonic Hedgehog in Floor Plate and Notochord abstract: The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish. We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in intron 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse. Functional analysis in the zebrafish embryo revealed that the intronic enhancers have a complex organisation. Two activator regions, ar- A and ar-C, were identified in intron 1 and 2, respectively, which mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three regions of 40 bp to be essential for activity. These regions do not contain binding sites for HNF 3beta, the winged helix transcription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcription factor-binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord- deficient mutant embryos.

A Novel Peptide Specifically Binding to Nasopharyngeal Carcinoma for Targeted Drug Delivery

title: A Novel Peptide Specifically Binding to Nasopharyngeal Carcinoma for Targeted Drug Delivery abstract: Nasopharyngeal carcinoma (NPC) is a common cancer among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate in the early stage of NPC has been reported as high as 90 to 95% with the use of radiotherapy, but in the advanced cases, even with the use of both chemotherapy and radiotherapy, the survival rate is still < 50%. To improve the survival rate, we identify a 12-mer peptide (L-peptide) specifically binding to NPC cells with a phage displayed random peptide library. The L-pliage and synthetic L-peptide bound to the tumor cell surfaces of most NPC cell lines and biopsy specimens, but not normal nasal mucosal cells, and the L-peptide-linked liposomes containing fluorescent substance (L-peptide-Lipo-HPTS) were capable of binding to and translocating across plasma membranes. L- Peptide-linked liposomes that carried doxorubicin (L-peptide -Lipo-Dox) caused marked cytotoxicity in NPC cells. In SCID mice bearing NPC xenografts, the L-phages specifically bound to the tumor mass, an effect that was inhibited by competition with synthetic L-peptide. In addition, the L- peptide-Lipo-Dox suppressed tumor growth better than Lipo- Dox. These results indicate that the novel L- peptide specifically binds NPC cells and is a good candidate for targeted drug delivery to NPC solid tumors.

Mdm2 Expression in Ebv-Infected Nasopharyngeal Carcinoma Cells

title: Mdm2 Expression in Ebv-Infected Nasopharyngeal Carcinoma Cells abstract: To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC -TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV- infected (EBV+) cells. In the animals bearing EBV and EBV- NPC xenografts, the MDM2(+) cells only appeared in clusters in both EBV+ and EBV- tumors with stronger expression in EBV cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV -LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2 -Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2 -Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm 2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis .

A Possible Molecular Mechanism of Hanatoxin Binding-Modified Gating in Voltage-Gated K+-Channels

title: A Possible Molecular Mechanism of Hanatoxin Binding-Modified Gating in Voltage-Gated K+-Channels abstract: While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interest concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated . Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and further experimental designs (Lou et al., 2002. J. Mol. Recognit. 15: 175-179). However, if one only relies on such information, more advanced structure-functional interpretations for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unknown. In order to have better understanding of the molecular details regarding this issue, we have performed the docking simulation with the S3 C sequence from the hanatoxin-insensitive K+-channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of the S3-S4 linker region, we suggested a significant movement of drk1 S3 C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering with S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of the S3-S4 linker, a possible molecular mechanism to illustrate the hanatoxin binding- modified gating is proposed. Copyright 2003 John Wiley Sons, Ltd.