Cheuk, Wai Ka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 96-108). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xii / Chapter CHAPTER 1 --- General introduction / Chapter 1.1 --- Metal Contaminations in the environment --- p.1 / Chapter 1.2 --- Biology of Heavy Metal Ions --- p.3 / Chapter 1.2.1 --- Essential and non-essential metal ions --- p.3 / Chapter 1.2.2 --- Toxicities and origins of heavy metal ions --- p.5 / Chapter 1.3 --- Monitoring Of Heavy Metal Contaminations In Aquatic Environment --- p.9 / Chapter 1.3.1 --- Monitoring in chemical approach --- p.9 / Chapter 1.3.2 --- Monitoring in biological approach: biomarkers --- p.11 / Chapter 1.4 --- Metallothionein (MT) --- p.12 / Chapter 1.4.1 --- Biological functions of MT and its regulation --- p.12 / Chapter 1.4.2 --- MT isoforms --- p.14 / Chapter 1.4.3 --- Mechanisms of MT gene regulation --- p.15 / Chapter 1.4.3.1 --- Zinc pool hypothesis --- p.20 / Chapter 1.4.3.2 --- Protein kinase cascade --- p.21 / Chapter 1.5 --- Metal responsive element (MRE) --- p.22 / Chapter 1.6 --- MRE-Binding Transcription Factor-1 (MTF-1) --- p.30 / Chapter 1.6.1 --- Structure of MTF-1 --- p.30 / Chapter 1.6.2 --- Physiological functions of MTF-1 --- p.32 / Chapter 1.6.3 --- The role of MTF-1 in MT gene regulation --- p.33 / Chapter 1.6.4 --- Regulation of MTF-1 by various heavy metals --- p.34 / Chapter 1.7 --- Zebrafish (Daino reio) --- p.36 / Chapter 1.8 --- Project aim --- p.37 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Cell Culture --- p.40 / Chapter 2.1.1 --- ZFL cell line --- p.40 / Chapter 2.1.2 --- SJD cell line --- p.41 / Chapter 2.2 --- Alarmar blue̐ưؤ M assay --- p.41 / Chapter 2.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.1 --- Metal treatment of the SJD and ZFL cell lines --- p.42 / Chapter 2.3.2 --- Isolation of total RNA --- p.43 / Chapter 2.3.3 --- Quantification of mRNA by spectrophotometer --- p.43 / Chapter 2.3.4 --- Reverse Transcription --- p.44 / Chapter 2.4 --- Quantifications of mRNA levels by using real-time PCR technique --- p.44 / Chapter 2.4.1 --- Primer design --- p.44 / Chapter 2.4.2 --- PCR components and cycling condition --- p.45 / Chapter 2.4.3 --- Determination of relative amount of target gene present in the samples --- p.49 / Chapter 2.5 --- Cloning of zMT-II gene promoter and its transient expression studies --- p.50 / Chapter 2.5.1 --- Purification of genomic DNA --- p.50 / Chapter 2.5.2 --- Preparation of Escherichia coli competent cell --- p.51 / Chapter 2.5.3 --- PCR-Cloning of a 1.4 kb zMT-II gene promoter --- p.51 / Chapter 2.5.4 --- Purification of plasmid DNA --- p.53 / Chapter 2.5.5 --- Transient transfection of plasmid into SJD and ZFL cells --- p.54 / Chapter 2.5.6 --- Heavy metal treatments and measurement of luciferase activities --- p.54 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Toxicities of various heavy metal ions --- p.56 / Chapter 3.2 --- Relative mRNA fold induction of zMT in SJD and ZFL cell lines --- p.59 / Chapter 3.3 --- The zMT-II gene and its induction by metal ions in zebrafish cell-lines --- p.63 / Chapter 3.4 --- MTF-1 mRNA levels in SJD and ZFL cell lines exposed to heavy metal ions --- p.74 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Comparison of metal toxicities in the two cell lines studied --- p.78 / Chapter 4.2 --- zMT gene expression study --- p.80 / Chapter 4.2.1 --- zMT mRNA regulation by heavy metal ions in the two cell lines --- p.80 / Chapter 4.2.2 --- The potential use of MT regulation as exposure biomarker --- p.82 / Chapter 4.3 --- Structure of the zMT-II gene promoter region --- p.82 / Chapter 4.4 --- Metal responsiveness of zMT-II promoter --- p.84 / Chapter 4.5 --- Mechanism of MT gene expression and the MTF-1 mRNA inductions in SJD and ZFL cell lines --- p.86 / Chapter 4.6 --- Concluding Remarks --- p.93 / References --- p.96

Mast cells play pivotal roles in immunoglobulin (Ig) E-mediated airway inflammation, expressing interleukin (IL)-13 and monocyte chemoattractant protein-1 (MCP-1), which in turn regulate IgE synthesis and/or inflammatory cell recruitment. The molecular effects of IL-1beta on cytokine expression by human mast cells (HMC) have not been studied well. In this report, we provide evidence that human umbilical cord blood-derived mast cells (CBDMC) and HMC-1 cells express the type 1 receptor for IL-1. We also demonstrate that IL-1beta and tumor necrosis factor-alpha are able to induce, individually or additively, dose-dependent expression of IL-13 and MCP-1 in these cells. The induction of IL-13 and MCP-1 gene expression by IL-1beta was accompanied by the activation of IL-1 receptor-associated kinase and translocation of the transcription factor, nuclear factor (NF) kappaB into the nucleus. Accordingly, Bay-11 7082, an inhibitor of NF-kappaB activation, inhibited IL-1beta-induced IL-13 and MCP-1 expression. IL-1beta also induced IL-13 promoter activity while enhancing the stability of IL-13 messenger RNA transcripts. Dexamethasone, a glucocorticoid, inhibited IL-1beta-induced nuclear translocation of NF-kappaB and also the secretion of IL-13 from mast cells. Our data suggest that IL-1beta can serve as a pivotal costimulus of inflammatory cytokine synthesis in human mast cells, and this may be partly mediated by IL-1 receptor-binding and subsequent signaling via nuclear translocation of NF-kappaB. Because IL-1beta is a ubiquitously expressed cytokine, these findings have important implications for non-IgE-mediated signaling in airway mast cells as well as for innate immunity and airway inflammatory responses, such as observed in extrinsic and intrinsic asthma.