The nucleus may be the critical site for the activation of chemical carcinogens, and subsequently the initiation of neoplasia. However, isolated nuclei may be contaminated with endoplasmic reticulum, the major site of the drug metabolizing enzymes. One of the objectives of the present study was to determine whether the enzymes in isolated rat hepatic nuclei were of nuclear origin and, if so, to compare these enzymes with those in the microsomal fraction. The selective manipulation of nuclear enzymes would be a useful tool in determining their role in cellular toxicity. Recentrifugation experiments, with aryl hydrocarbon hydroxylase (AHH) activity as a marker, showed that isolated nuclei were not contaminated with endoplasmic reticulum in the form of microsomes formed upon homogenization. However, small "tags" of endoplasmic reticulum, continuous with the nuclear membrane, and indiscernable in electron micrographs, could remain following centrifugation and account for all of the measurable enzyme activity in the isolated nuclei. It was reasoned that if endoplasmic reticulum accounted for all of the activity, then the ratio of nuclear to microsomal activity for all enzymes determined should be the same. The ratios of epoxide hydrolase and AHH were found to differ in the two fractions. The simplest interpretation of these data was that drug metabolizing enzymes existed in the nuclei. However, the distribution of drug metabolizing enzymes throughout the endoplasmic reticulum is known to be heterogeneous and these "tags" could differ from the total endoplasmic reticulum (microsomes) in their enzyme make-up. Whether these enzymes are in the nuclear membrane, nucleoplasm, or as "tags" of endoplasmic reticulum, they represent activities in close proximity to potential target sites in the nucleus. The inhibition, induction, and activation characteristics of nuclear and microsomal enzymes were studied with the goal of selective manipulation of nuclear enzymes. The enzymes in the nuclear and microsomal fractions were found to differ' only in quantitative inducibility, and were identical in all other respects. Therefore, the selective manipulation of nuclear enzymes was not achieved. The induction of hepatic drug metabolizing enzymes is a measure of altered genetic expression in the liver. Inducers of drug metabolizing enzymes have also been shown to promote neoplasia in the liver. Therefore, studying the induction of such enzymes may lead to a further understanding of the mechanism of tumour promotion. Phenobarbital, 3-methylcholanthrene and pregnenolone-16α-carbonitrile produce three distinct induction responses. In the present study, spironolactone and trans-stiIbene oxide were shown to produce distinct induction responses, also. Spironolactone was shown to be a different inducer based on the protein band patterns observed following SDS-polyacrylamide gel electrophoresis of liver microsomes. trans-Stilbene oxide was found to produce a significantly different maximal level of AHH activity. The observation of five distinct induction responses suggests at least five recognition sites (receptors) mediating the pleiotropic actions of exogenous compounds in the liver. / Pharmaceutical Sciences, Faculty of / Graduate

The metabolism of lignocaine to monoethylglycinexylidide has been found useful as an indicator of hepatic function in association with liver transplantation. It has been postulated that this might be due to the common effect of hypoxic damage on liver function and lignocaine metabolism. The aim of this work was to establish whether hepatic lignocaine elimination was impaired by hypoxia and whether lignocaine extraction ratio and clearance could be used as an indicator of hepatic function. This was studied using the isolated pig liver perfused via the hepatic artery and portal vein. To establish whether the pig liver could be used as a possible human model for this investigation and whether lignocaine had any detrimental effects on liver function and blood flow in vivo, hepatic lignocaine elimination and the effects of lignocaine administration on hepatic function and blood flow were studied in the anaesthetized pig, surgically prepared to allow sampling across the liver and direct hepatic blood flow measurement. Hepatic lignocaine elimination was then studied in the isolated perfused liver to determine whether this was similar to that found in vivo. The definitive studies required preliminary investigations not available from the literature to determine the feasibility of comparing in vivo and ex vivo hepatic function using the same liver. In addition, by studying the decay of lignocaine after bolus dose administration the necessary pharmacokinetic parameters to achieve similar constant hepatic affluent lignocaine concentrations in vivo and in the isolated preparation could be determined. The preliminary investigations showed that a sequential experiment using the same liver to compare in vivo and ex vivo function was inappropriate as the energy state of isolated perfused livers previously studied in vivo was significantly different from that in livers perfused immediately. The decay of lignocaine after a bolus dose in vivo and ex vivo could be described by a two-compartment open model and in both preparations the derived pharmacokinetic parameters from this analysis were used to achieve similar constant hepatic affluent concentrations over the study period used to determine hepatic lignocaine elimination. Lignocaine extraction ratio by the in situ pig liver was similar to that reported in man and together with hepatic clearance and intrinsic clearance was similar to that determined in the isolated state when different livers were used for this comparison. There was no detrimental effect of lignocaine administration on hepatic function and blood flow In vivo. Lignocaine extraction ratio and clearance and monoethylglycinexylidide formation were significantly impaired in livers subjected to hypoxia. Lignocaine elimination correlated strongly with hepatic cellular ATP, energy charge and ATP/ ADP ratio as well as with hepatic potassium release but less strongly with aspartate aminotransferase release when this relationship was tested using the combined data from hypoxic and normoxic livers ex vivo. These correlations were positive for hepatic adenine nucleotide status and negative for hepatic potassium and aspartate aminotransferase release. Neither hepatic alanine aminotransferase release nor lactate utilization were significantly affected by hypoxia. Lignocaine extraction ratio, hepatic oxygen consumption, ATP content, bile flow and potassium release were shown to be equivalent, more highly sensitive, and earlier indicators of hypoxic hepatic injury than hepatic aspartate aminotransferase release in the isolated perfused pig liver.

The aims of the research were to establish whether diphenylhydantoin and phenobarbitone are teratogenic in mice both in vivo and in an in vitro whole embryo culture system, to investigate possible mechanisms of teratogenicity and to examine whether the methods used in this study, may form a basis for developing systems of more extensive drug teratogenicity screening.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow-derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. β-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic β-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that β-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

Po Lai See. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-152). / Abstracts in English and Chinese. / TITLE PAGE --- p.p.1 / ACKNOWLEGDEMENTS --- p.p.2 / ABSTRACT --- p.p.3 / 摘要 --- p.p.6 / TABLE OF CONTENTS --- p.p.9 / LIST OF FIGURES AND TABLES --- p.p.16 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Estrogen and Estrogen Receptors and its Action --- p.p.18 / Chapter 1.1.1 --- Estrogen --- p.p.19 / Chapter 1.1.2 --- Estrogen Receptors --- p.p.19 / Chapter 1.1.3 --- Structural Differences between ERa and ERp --- p.p.21 / Chapter 1.1.4 --- Functional Differences --- p.p.22 / Chapter 1.1.5 --- Effects of Selective Estrogen Receptor Modulators --- p.p.22 / Chapter 1.1.6 --- Estrogen works --- p.p.23 / Chapter 1.1.7 --- Estrogen Receptors and Breast Cancer --- p.p.24 / Chapter 1.2 --- Flavonoids： Properties and Biological Activities --- p.p.25 / Chapter 1.2.1 --- Chemical Structure and Classification of flavonoids --- p.p.25 / Chapter 1.2.2 --- Biological Properties and Action Mechanism of Flavonoids… --- p.p.27 / Chapter 1.2.3 --- Flavonoids and breast cancer prevention --- p.p.27 / Chapter 1.3 --- Aims and Scopes of Investigation --- p.p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.p.30 / Chapter 2.1.1 --- Flavonoids --- p.p.30 / Chapter 2.1.2 --- Plasmids --- p.p.30 / Chapter 2.2 --- Mammalian cell culture --- p.p.31 / Chapter 2.2.1 --- Maintenance of cells --- p.p.31 / Chapter 2.2.2 --- Preparation of cell stock --- p.p.32 / Chapter 2.2.3 --- Cell recovery from liquid nitrogen stock --- p.p.32 / Chapter 2.3 --- Identification of estrogenic activity in flavonoids --- p.p.33 / Chapter 2.3.1 --- Steady Glo Luciferase Assay --- p.p.33 / Chapter 2.3.2 --- The Biorad Protein Assay kit (a modified Bradford method). --- p.p.33 / Chapter 2.4 --- Viability Assay --- p.p.34 / Chapter 2.5 --- ERE Luciferase reporter gene assay --- p.p.35 / Chapter 2.5.1 --- Transient transfect ion of cell using lipofectamine PLUS reagent --- p.p.36 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.p.37 / Chapter 2.6 --- ERα competitive binding ASSAY --- p.p.37 / Chapter 2.7 --- Apoptotic death assay --- p.p.38 / Chapter 2.8 --- Semi-quantitative RT-PCR Assay --- p.p.40 / Chapter 2.8.1 --- "Isolation of RNA using TRIzol® Reagent (Life Technology,USA) " --- p.p.40 / Chapter 2.8.2 --- Quantitation of RNA --- p.p.41 / Chapter 2.8.3 --- First strand cDNA synthesis --- p.p.41 / Chapter 2.8.4 --- PCR reactions --- p.p.43 / Chapter 2.9 --- Flow Cytometry Analysis --- p.p.43 / Chapter 2.10 --- Total triglyceride and cholesterol measurement --- p.p.44 / Chapter 2.10.1 --- Determination of the total cholesterol --- p.p.45 / Chapter 2.10.2 --- Determination of the total triglyceride --- p.p.46 / Chapter 2.11 --- Manipulation of DNA and RNA --- p.p.46 / Chapter 2.11.1 --- Transformation of DH5α --- p.p.46 / Chapter 2.11.2 --- Mini preparation of plasmid DNA --- p.p.47 / Chapter 2.11.3 --- Preparation of plasmid DNA using QIAGEN-tip 100 midi-prep kit --- p.p.48 / Chapter 2.11.4 --- Preparation of plasmid DNA using QIAGEN-tip 10000 Giga-prep kit --- p.p.49 / Chapter 2.11.5 --- Ethanol preparation of DNA and RNA --- p.p.50 / Chapter 2.11.6 --- Agarose gel electrophoresis of DNA --- p.p.51 / Chapter 2.12 --- Statistical methods --- p.p.52 / Chapter CHAPTER 3 --- Estrogenic and antiproliferative activities on MCF-7 breast cancer cells by flavonoids / Chapter 3.1 --- Introduction --- p.p.53 / Chapter 3.2 --- Results --- p.p.56 / Screening of phytoestrogens for estrogenic activities on MELN cells --- p.p.56 / Cell proliferation activity of phytoestrogens on MCF-7 and MDA-MA231 cells --- p.p.59 / Estrogenic and antiestrogenic activity of phytoestrogens on ERα or erβ transfected hepg2 cells --- p.p.64 / Chapter 3.3 --- Discussion --- p.p.73 / Chapter Chapter 4 --- interaction of baicalein with estrogen receptors / Chapter 4.1 --- Introduction --- p.p.76 / Chapter 4.2 --- Results --- p.p.78 / Estrogen receptor competition assay --- p.p.78 / ERE-Luciferase gene reporter assay --- p.p.82 / Chapter 4.3 --- Discussion --- p.p.88 / Chapter Chapter 5 --- baicalein and genistein display differential actions on er transactivation / Chapter 5.1 --- Introduction --- p.p.90 / Chapter 5.2 --- Results --- p.p.92 / Estrogenic and antiestrogenic activities of genistein and baicalein on ER transactivation --- p.p.92 / Chapter 5.3 --- Discussion --- p.p.105 / Chapter CHAPTER 6 --- APOPTOTIC EFFECTS OF BAICALEIN ON MCF-7 AND MDA-MB-231 CELL LINES / Chapter 6.1 --- Introduction --- p.p.107 / Chapter 6.2 --- Results --- p.p.111 / ER POSITIVE MCF-7 AND ER NEGATIVE MDA-MB-231 cell death assay --- p.p.111 / "Bcl-2, Bax and PS2 mRNA expression " --- p.p.116 / Arrest at sub G1 phase of MCF-7 by baicalein --- p.p.124 / Chapter 6.3 --- Discussion --- p.p.127 / Chapter CHAPTER 7 --- BAICALEIN CAN REDUCE INTRACELLULAR cholesterol and triglceride / Chapter 5.1 --- Introduction --- p.p.129 / Chapter 5.2 --- Results --- p.p.130 / Baicalein has beneficial effect on lipid metabolism --- p.p.130 / Chapter 5.3 --- Discussion --- p.p.139 / Chapter chapter 8 --- Summary --- p.p.140 / BIBLIOGRAPHY --- p.p.142 / APPENDIX 1 ABBREVIATIONS --- p.p.153 / APPENDIX 2 PRIMER LISTS --- p.p.156 / APPENDIX 3 REAGENTS AND BUFFERS --- p.p.157

Chan Yau Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 157-177). / Abstracts in English and Chinese. / Contents / Declaration --- p.i / Acknowledgement --- p.ii / Abbreviations --- p.iii-iv / Abstract in English --- p.v-viii / Abstract in Chinese --- p.ix-xi / Contents --- p.xii-xvi / Chapter CHAPTER I - --- Introduction / Chapter 1.1. --- Selective Estrogen Receptor Modulators (SERMs) --- p.1 / Chapter 1.1.1. --- Raloxifene --- p.6 / Chapter 1.2. --- Mechanisms of Action of SERMs in Vascular System --- p.7 / Chapter 1.2.1. --- Estrogen --- p.7 / Chapter 1.2.2. --- Estrogen Receptors (ERs) --- p.8 / Chapter 1.2.3. --- General Mechanisms of Action of SERMs --- p.13 / Chapter 1.2.4. --- Actions of Raloxifene --- p.14 / Chapter 1.3. --- Effects of SERMs in Cardiovascular System --- p.14 / Chapter 1.3.1. --- Effects of SERMs on Endothelial Function --- p.15 / Chapter 1.3.2. --- Effects of SERMs on Vascular Smooth Muscle --- p.17 / Chapter 1.4. --- Effects of Raloxifene on Vascular Circulations --- p.18 / Chapter 1.4.1. --- Effects of Raloxifene on Systemic Circulation --- p.18 / Chapter 1.4.1.1. --- Preclinical Data --- p.18 / Chapter 1.4.1.1.1. --- Effects on Serum Lipids --- p.18 / Chapter 1.4.1.1.2. --- Effects on Inflammation Markers and Blood Coagulation --- p.19 / Chapter 1.4.1.1.3. --- Antioxidative Effects --- p.19 / Chapter 1.4.1.1.4. --- Effects on Nitric Oxide and Endothelial Function --- p.19 / Chapter 1.4.1.1.5. --- Effects on Vascular Smooth Muscle --- p.20 / Chapter 1.4.1.1.6. --- "Vascular Injury, Atherosclerosis and Ischaemia-Reperfusion Injury" --- p.20 / Chapter 1.4.1.2. --- Clinical Studies - Effects in Post-Menopausal Women --- p.21 / Chapter 1.4.1.2.1. --- "Effects on Serum Lipids, Lipoproteins and Triglycerides" --- p.21 / Chapter 1.4.1.2.2. --- Effects on Inflammation Markers and Homocysteine --- p.22 / Chapter 1.4.1.2.3. --- Effects on Coagulation Markers --- p.23 / Chapter 1.4.1.2.4. --- Effects on Endothelial Function --- p.23 / Chapter 1.4.1.2.5. --- Cardiovascular Events --- p.23 / Chapter 1.5. --- Myogenic Response and Vascular System --- p.24 / Chapter 1.5.1. --- Initiation and Development of Myogenic Response --- p.25 / Chapter 1.5.2. --- Regulation of Myogenic Response --- p.26 / Chapter 1.5.2.1. --- 20-hydroxyeicosatetraenoic acid (20-HETE) --- p.26 / Chapter 1.5.2.2. --- "Protein Kinase C, Rho/Rho-Kinase, and Tyrosine Kinase" --- p.27 / Chapter 1.5.3. --- Myogenic Response and Endothelium --- p.31 / Chapter 1.5.4. --- Estrogen and Myogenic Tone --- p.31 / Chapter 1.6. --- Objectives of the Present Study --- p.32 / Chapter CHAPTER II - --- Methods and Materials / Chapter 2.1. --- Tissue and Cell Preparation --- p.34 / Chapter 2.1.1. --- Vessel Preparation --- p.34 / Chapter 2.1.2. --- Removal of a Functional Endothelium --- p.36 / Chapter 2.2. --- Myograph and Pressure Myograph Setups --- p.36 / Chapter 2.2.1. --- Myograph 一 Isometric Tension Measurement --- p.36 / Chapter 2.2.2. --- Pressure Myograph - Isobaric Diameter Measurement --- p.37 / Chapter 2.3. --- Intracellular [Ca2+] Measurement in Vascular Smooth Muscle --- p.42 / Chapter 2.4. --- Chronic Raloxifene Therapyin Spontaneously Hypertensive Rats (SHRs) and Wistar-Kyoto Rats (WKYs) --- p.42 / Chapter 2.4.1. --- Surgical Procedure - Raloxifene Tubing Insertion --- p.42 / Chapter 2.4.2. --- "Body Weight, Mean Arterial Blood Pressure and Uterine Weight" --- p.42 / Chapter 2.4.3. --- Measurement of Raloxifene Tubing Consumption --- p.43 / Chapter 2.4.4. --- Effect of Chronic Raloxifene Treatment on Artery Reactivity --- p.43 / Chapter 2.5. --- Ovariectomy and Chronic Raloxifene Therapyin Syrian Golden Hamsters --- p.45 / Chapter 2.5.1. --- Surgical Procedure - Ovariectomy (OVX) --- p.45 / Chapter 2.5.2. --- Surgical Procedure - Raloxifene Tubing Insertion --- p.45 / Chapter 2.5.3. --- High-Cholesterol Food Preparation --- p.45 / Chapter 2.5.4. --- "Body Weight, Food Consumption and Uterine Weight" --- p.46 / Chapter 2.5.5. --- Measurement of Raloxifene Tubing Consumption --- p.46 / Chapter 2.5.6. --- Serum Lipid and Lipoprotein Determinations --- p.46 / Chapter 2.5.7. --- Effect of Chronic Raloxifene on Artery Reactivity --- p.46 / Chapter 2.6. --- Solutions and Drugs --- p.49 / Chapter 2.6.1. --- "Drugs, Chemicals and Enzymes" --- p.49 / Chapter 2.6.2. --- Solutions --- p.51 / Chapter 2.6.3. --- Diet Composition for Syrian Golden Hamsters --- p.51 / Chapter 2.7. --- Statistical Analysis --- p.52 / Chapter CHAPTER III - --- "Raloxifene Relaxes Rat Pulmonary Arteries and Veins: Roles of Gender, Endothelium, and Antagonism of Ca Influx" / Chapter 3.1. --- Abstract --- p.53 / Chapter 3.2. --- Introduction --- p.54 / Chapter 3.3. --- Methods and Materials --- p.55 / Chapter 3.3.1. --- Blood Vessel Preparation --- p.55 / Chapter 3.3.2. --- Protocols --- p.55 / Chapter 3.3.3. --- Measurement of Vascular Smooth Muscle [Ca2+]i --- p.56 / Chapter 3.3.4. --- Drugs --- p.57 / Chapter 3.3.5. --- Data Analysis --- p.53 / Chapter 3.4. --- Results --- p.58 / Chapter 3.4.1. --- Effects of Raloxifene on Pulmonary Arteries --- p.53 / Chapter 3.4.2. --- Effect of Raloxifene on CaCl2-induced Constrictionin Pulmonary Arteries --- p.59 / Chapter 3.4.3. --- Effects of Raloxifene on Pulmonary Veins --- p.59 / Chapter 3.4.4. --- Effect of Raloxifene on CaCl2-stimulated Increases in [Ca2+]i in Pulmonary Arteries --- p.60 / Chapter 3.5. --- Discussion --- p.67 / Chapter 3.6. --- Conclusion --- p.69 / Chapter CHAPTER IV - --- Raloxifene Modulates Pulmonary Vascular Reactivity in Spontaneously Hypertensive Rats / Chapter 4.1. --- Abstract --- p.70 / Chapter 4.2. --- Introduction --- p.71 / Chapter 4.3. --- Methods and Materials --- p.72 / Chapter 4.3.1. --- Raloxifene Treatment --- p.72 / Chapter 4.3.2. --- Blood Vessel Preparation --- p.72 / Chapter 4.3.3. --- Protocols --- p.73 / Chapter 4.3.4. --- Chemicals and Drugs --- p.73 / Chapter 4.3.5. --- Data Analysis --- p.74 / Chapter 4.4. --- Results --- p.74 / Chapter 4.4.1. --- Blood Pressure --- p.74 / Chapter 4.4.2. --- Vasocontraction in Spontaneously Hypertensive Rats --- p.75 / Chapter 4.4.3. --- Vasorelaxation in Spontaneously Hypertensive Rats --- p.75 / Chapter 4.4.4. --- Vasocontraction in Wistar-Kyoto rats --- p.76 / Chapter 4.4.5. --- Vasorelaxation in Wistar-Kyoto rats --- p.77 / Chapter 4.4.6. --- Comparison of contraction between WKY and SHR rats --- p.78 / Chapter 4.4.7. --- Comparison of relaxation between WKY and SHR rats --- p.78 / Chapter 4.5. --- Discussion --- p.93 / Chapter 4.6. --- Conclusion --- p.96 / Chapter CHAPTER V - --- Effects of Therapeutic Concentrations of Raloxifene in Pressurized Rat Small Mesenteric Artery / Chapter 5.1. --- Abstract --- p.98 / Chapter 5.2. --- Introduction --- p.99 / Chapter 5.3. --- Methods and Materials --- p.101 / Chapter 5.3.1. --- Blood Vessel Preparation --- p.101 / Chapter 5.3.2. --- Experimental Protocols --- p.102 / Chapter 5.3.2.1. --- Myogenic Tone Development --- p.102 / Chapter 5.3.2.2. --- Effects of Raloxifene and 17β-EstradioI on Myogenic Constriction --- p.102 / Chapter 5.3.2.3. --- Effects of Pharmacological Inhibitors on Raloxifene- or 17β-Estradiol-induced Myogenic Constriction --- p.103 / Chapter 5.3.3. --- Drugs and Solutions --- p.103 / Chapter 5.3.4. --- Expression of Results and Statistical Analysis --- p.104 / Chapter 5.4. --- Results --- p.104 / Chapter 5.4.1. --- Effects of Raloxifene and 17β-Estradiol on Rat Resistance Mesenteric Arteries1 --- p.104 / Chapter 5.4.2. --- Effects of Inhibitors of NOS --- p.105 / Chapter 5.4.3. --- Effect of CTX plus Apamin --- p.106 / Chapter 5.4.4. --- "Effect of ICI 182,780" --- p.106 / Chapter 5.4.5. --- "Effects of Wortmannin, LY 294002 and Cycloheximide" --- p.106 / Chapter 5.5. --- Discussion --- p.122 / Chapter 5.6. --- Conclusion --- p.125 / Chapter CHAPTER VI - --- Effects of Chronic Raloxifene Treatment on Vascular Reactivity in Pressurized Septal Coronary Arteries from Hamsters Fed with High-Cholesterol Diet / Chapter 6.1. --- Abstract --- p.127 / Chapter 6.2. --- Introduction --- p.128 / Chapter 6.3. --- Methods and Materials --- p.129 / Chapter 6.3.1. --- Preparatory Work --- p.129 / Chapter 6.3.1.1. --- Animals and Diets --- p.129 / Chapter 6.3.1.2. --- Preparation of High-Cholesterol (HC) Food --- p.129 / Chapter 6.3.1.3. --- Surgical Procedure - Ovariectomy (OVX) --- p.129 / Chapter 6.3.1.4. --- Surgical Procedure - Raloxifene Tubing Insertion --- p.130 / Chapter 6.3.1.5. --- Blood Vessel Preparation --- p.130 / Chapter 6.3.1.6. --- "Body Weight, Food Consumption and Uterine Weight" --- p.131 / Chapter 6.3.1.7. --- Measurement of Raloxifene Tubing Consumption --- p.131 / Chapter 6.3.1.8. --- Serum Lipid and Lipoprotein Determinations --- p.132 / Chapter 6.3.2. --- Experimental Protocols --- p.132 / Chapter 6.3.2.1. --- Development of Myogenic Tone --- p.132 / Chapter 6.3.2.2. --- Pressure-Diameter Relationships --- p.132 / Chapter 6.3.2.3. --- The Effect of Acetylcholine --- p.133 / Chapter 6.3.2.4. --- The Effect of U46619 --- p.133 / Chapter 6.3.2.5. --- The Effect of L-NAME --- p.133 / Chapter 6.3.3. --- Drugs and Solutions --- p.133 / Chapter 6.3.4. --- Expression of Results and Statistical Analysis --- p.134 / Chapter 6.4. --- Results --- p.135 / Chapter 6.4.1. --- Effects on Myogenic Response --- p.135 / Chapter 6.4.2. --- "Effects of Acetylcholine, U46619 and L-NAME" --- p.135 / Chapter 6.4.2.1. --- Comparison between OHHCD and OvxOHHCD --- p.135 / Chapter 6.4.2.2. --- Comparison between OvxOHHCD and OvxOHHCDRf --- p.135 / Chapter 6.4.2.3. --- Comparison between OHHCDRf and OvxOHHCDRf --- p.136 / Chapter 6.4.2.4. --- Comparison between OHHCD and OHHCDRf --- p.136 / Chapter 6.5. --- Discussion --- p.155 / Chapter 6.6. --- Conclusion --- p.156 / References --- p.157 / Publications --- p.176

Kam Man Fai Afia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [195]-224). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The Mast Cell --- p.2 / Chapter 1.1.1 --- Historical Perspective --- p.2 / Chapter 1.1.2 --- Mast Cell Origin and Development --- p.3 / Chapter 1.1.3 --- Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.1 --- Rodent Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.2 --- Human Mast Cell Heterogeneity --- p.7 / Chapter 1.1.4 --- Mast Cell Mediators --- p.10 / Chapter 1.1.4.1 --- Preformed Mediators --- p.11 / Chapter 1.1.4.2 --- Newly Synthesized Lipid Mediators --- p.14 / Chapter 1.1.4.3 --- Cytokines --- p.16 / Chapter 1.1.5 --- Mast Cell Activation --- p.17 / Chapter 1.1.5.1 --- Immunological Activation --- p.19 / Chapter 1.1.5.1.1 --- FcεIR Activation and Protein Tyrosine Phosphorylation --- p.19 / Chapter 1.1.5.1.2 --- Activation of Phospholipases --- p.20 / Chapter 1.1.5.1.3 --- The Role of Calcium --- p.22 / Chapter 1.1.5.1.3.1 --- Intracellular Calcium Mobilization --- p.23 / Chapter 1.1.5.1.3.2 --- Calcium Influx --- p.24 / Chapter 1.1.5.1.3.3 --- Mechanisms of Action of Calcium in Mast Cells --- p.28 / Chapter 1.1.5.1.4 --- The Role of G-proteins --- p.30 / Chapter 1.1.5.1.5. --- The Role of Cylic AMP --- p.33 / Chapter 1.1.5.1.2.1 --- Mechanisms of Action of Cyclic AMP in Mast Cells --- p.36 / Chapter 1.1.5.1.2.2 --- Implications for the Inhibitory Role of Cyclic AMP in Mast Cell Activation --- p.37 / Chapter 1.2 --- The Cyclic Nucleotide Phosphodiesterases --- p.39 / Chapter 1.2.1 --- Introduction --- p.39 / Chapter 1.2.2 --- Classification and Structure --- p.41 / Chapter 1.2.3 --- Distribution and Physiological Functions of the Different PDE Families --- p.45 / Chapter 1.2.4 --- Phosphodiesterase Inhibitors --- p.49 / Chapter 1.2.4.1 --- Non-selective PDE Inhibitors --- p.50 / Chapter 1.2.4.2 --- Selective PDE Inhibitors --- p.52 / Chapter 1.2.4.2.1 --- PDE1 and PDE2 Inhibitors --- p.52 / Chapter 1.2.4.2.2 --- PDE3 Inhibitors --- p.53 / Chapter 1.2.4.2.3 --- PDE4 Inhibitors --- p.54 / Chapter 1.2.4.2.4.1 --- PDE5 Inhibitors --- p.56 / Chapter 2. --- Materials and Methods --- p.59 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Drugs --- p.60 / Chapter 2.1.1.1 --- Phosphodiesterase Inhibitors --- p.60 / Chapter 2.1.1.2 --- Mast Cell Secretagogues --- p.61 / Chapter 2.1.2 --- Materials for Rat Peritoneal Mast Cell Experiments --- p.61 / Chapter 2.1.2.1 --- Materials for Rat Sensitization --- p.61 / Chapter 2.1.2.2 --- Materials for Buffers --- p.62 / Chapter 2.1.2.3 --- Materials for Histamine Assay --- p.62 / Chapter 2.1.2.4 --- Miscellaneous --- p.63 / Chapter 2.1.3 --- Materials for RBL-2H3 Cell Line Experiments --- p.63 / Chapter 2.1.3.1 --- Materials for Cell Culture --- p.63 / Chapter 2.1.3.2 --- Materials for Cell Sensitization and Enzyme Release --- p.64 / Chapter 2.1.3.3 --- Materials for β-Hexosaminidase Assay --- p.64 / Chapter 2.1.3.4 --- Miscellaneous --- p.64 / Chapter 2.2 --- Rat Peritoneal Mast Cell Experiments --- p.65 / Chapter 2.2.1 --- Preparation of Buffers --- p.65 / Chapter 2.2.2 --- Preparation of Stock Solutions --- p.66 / Chapter 2.2.2.1 --- Mast Cell Secretagogue Stock Solutions --- p.66 / Chapter 2.2.2.2 --- Phosphodiesterase Inhibitor Stock Solutions --- p.66 / Chapter 2.2.3 --- Animals and Cell Isolation --- p.71 / Chapter 2.2.3.1 --- Animals --- p.71 / Chapter 2.2.3.2 --- Sensitization of Animals --- p.71 / Chapter 2.2.3.3 --- Cell Isolation --- p.71 / Chapter 2.2.3.4 --- Cell Purification --- p.72 / Chapter 2.2.3.5 --- Determination of Cell Number and Viability --- p.73 / Chapter 2.2.4 --- General Protocol for Histamine Release and Histamine Measurement --- p.75 / Chapter 2.2.4.1 --- Histamine Release --- p.75 / Chapter 2.2.4.2 --- Spectrofluorometric Determination of Histamine Content --- p.76 / Chapter 2.2.4.2.1 --- Manual Histamine Assay --- p.76 / Chapter 2.2.4.2.2 --- Automated Histamine Assay --- p.78 / Chapter 2.2.4.3 --- Calculation of Histamine Levels --- p.78 / Chapter 2.2.4.4 --- Presentation and Statistics --- p.79 / Chapter 2.3 --- RBL-2H3 Cell Line Experiments --- p.80 / Chapter 2.3.1 --- Preparation of Stock Solutions --- p.80 / Chapter 2.3.2 --- Preparation of Materials for Enzyme Release and Assay --- p.81 / Chapter 2.3.2.1 --- Cell Culture --- p.81 / Chapter 2.3.2.2 --- Preparation of Cells for β-Hexosaminidase Release Experiments --- p.82 / Chapter 2.3.2.3 --- β-Hexosaminidase Release --- p.82 / Chapter 2.3.2.4 --- β-Hexosaminidase Assay --- p.83 / Chapter 3. --- Effects of Phosphodiesterase Inhibitors on Mediator Release from Rat Mast Cells --- p.84 / Chapter 3.1 --- Introduction --- p.85 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Rat Peritoneal Mast Cells --- p.87 / Chapter 3.2.1.1 --- Experiments Employing Immunological Stimulus in RPMCs --- p.87 / Chapter 3.2.1.2 --- Experiments Employing Non-Immunological Stimuli in RPMCs --- p.88 / Chapter 3.2.2 --- Rat Basophilic Leukemia Cells --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1 --- Immunologically Activated Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1.1 --- Effects of Non-Selective PDE Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.89 / Chapter 3.3.1.1.2 --- Effects of Selective PDE1 and PDE2 Inhibitors on Anti-IgE- Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.3 --- Effects of Selective PDE3 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.4 --- Effects of Selective PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.1.5 --- Effects of Selective PDE5 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.2 --- Non-Immunologically Activated Rat Peritoneal Mast Cells --- p.92 / Chapter 3.3.1.2.1 --- Effects of Selective PDE Inhibitors on Compound 48/80- Mediated Histamine Release from RPMCs --- p.92 / Chapter 3.3.1.2.2 --- Effects of Selective PDE Inhibitors on Histamine Release from RPMCs Stimulated by Calcium Ionophores --- p.93 / Chapter 3.3.2 --- Rat Basophilic Leukemia Cells --- p.93 / Chapter 3.3.2.1 --- Effects of Non-Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.93 / Chapter 3.3.2.2 --- Effects of Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.94 / Chapter 3.4 --- Discussion --- p.95 / Chapter 3.4.1 --- Rat Peritoneal Mast Cells --- p.95 / Chapter 3.4.1.1 --- Immunologically Activated RPMCs --- p.95 / Chapter 3.4.1.2 --- Non-Immunologically Activated RPMCs --- p.99 / Chapter 3.4.2 --- Rat Basophilic Leukemia Cells --- p.103 / Chapter 4. --- Combined Effects of Selective Phosphodiesterase Inhibitors on Immunologically Induced Histamine from Rat Mast Cells --- p.143 / Chapter 4.1 --- Introduction --- p.144 / Chapter 4.2 --- Materials and Methods --- p.144 / Chapter 4.2.1 --- Simultaneous Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.2.2 --- Sequential Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.3 --- Results --- p.146 / Chapter 4.3.1 --- Effects of the Selective Inhibitors for PDE3 and PDE4 Alone: Calculation of the Expected Inhibition Curve --- p.146 / Chapter 4.3.2 --- Effects of the Simultaneous Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.148 / Chapter 4.3.2.1 --- Rolipram and Siguazodan --- p.148 / Chapter 4.3.2.2 --- Ro 20-1724 and Siguazodan --- p.149 / Chapter 4.3.2.3 --- Rolipram and Quazinone --- p.149 / Chapter 4.3.2.4 --- Ro 20-1724 and Quazinone --- p.150 / Chapter 4.3.3 --- Effects of the Sequential Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.150 / Chapter 4.3.3.1 --- Rolipram and Siguazodan --- p.150 / Chapter 4.3.3.2 --- Ro 20-1724 and Siguazodan --- p.151 / Chapter 4.3.3.3 --- Rolipram and Quazinone --- p.151 / Chapter 4.3.3.4 --- Ro 20-1724 and Quazinone --- p.152 / Chapter 4.4 --- Discussion --- p.153 / Chapter 5. --- Future Directions --- p.191 / Chapter 5.1 --- Future Directions --- p.192 / References --- p.195

Hydroxyurea was used as a model teratogen to investigate the role of oxidative stress and stress-response pathways in mediating developmental toxicity. When administered to pregnant mice during early organogenesis, hydroxyurea induced fetal death and growth retardation, as well as external and skeletal malformations. The malformed fetuses displayed hindlimb, vertebral column, and tail defects. Hydroxyurea treatment enhanced the production of 4-hydroxynonenal, a lipid peroxidation end product, in malformation sensitive regions of the embryo. Depletion of glutathione, a major cellular antioxidant, specifically enhanced hydroxyurea-induced malformations and elevated the region-specific production of 4--hydroxynonenal protein adducts in the embryo, without affecting the incidence or extent of hydroxyurea-induced fetal death or growth retardation. The major proteins modified by 4-hydroxynonenal were involved in energy metabolism. Thus, oxidative stress is important in the induction of malformations by hydroxyurea. / Exposure to hydroxyurea stimulated the DNA binding activity of activator protein 1 (AP-1), an early response redox-sensitive transcription factor. Activated AP-1 was composed mainly of c-Fos heterodimers. Glutathione depletion did not change the effects of hydroxyurea on AP-1/c-Fos DNA binding activities despite an augmentation of the incidence of embryo malformations. Mitogen-activated protein kinases (MAPKs) activate AP-1 in response to stress by post-transcriptional phosphorylation of AP-1 proteins. Hydroxyurea treatment dramatically enhanced the activation of stress-responsive p38 MAPKs and JNKs (c-Jun N-terminal protein kinases). Selectively blocking p38 MAPKs enhanced the incidence of fetal death, whereas selective inhibition of JNKs specifically elevated the limb defects induced by hydroxyurea. Thus, activation of stress-response pathways impacts on the response of the embryo to a teratogenic insult.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.