by Kwan, Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 108-116). / Abstract --- p.i / Achnowledgment --- p.ii / List of abbreviations --- p.iii / List of table and figures --- p.v / Table of contents --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- THE DISCOVERY OF TUMOUR NECROSIS FACTOR (TNF) --- p.1 / Chapter 1.2 --- THE MOLECULE AND ITS RECEPTORS --- p.1 / Chapter 1.3 --- THE BIOLOGICAL ACTIVITIES OF TNF --- p.3 / Chapter 1.4 --- STUDIES ON THE CYTOTOXIC MECHANISM OF TNF --- p.4 / Chapter 1.5 --- A TENTATIVE MECHANISM OF TNF CYTOTOXICITY --- p.11 / Chapter 1.6 --- THE GLUTATHIONE SYSTEM : A CELLULAR PROTECTIVE MECHANISM AGAINST OXIDATIVE STRESS …… --- p.12 / Chapter 1.7 --- OBJECTIVE AND STRATEGY OF THIS STUDY --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND APPARATI / Chapter 2.1 --- CELL LINES --- p.19 / Chapter 2.2 --- "ISOLATION, MAINTENANCE AND SUBCULTURE OF CELL LINES" --- p.19 / Chapter 1. --- Plain RPMI-1640 medium / Chapter 2. --- Penicillin-streptomycin solution / Chapter 3. --- Foetal bovine serum / Chapter 4. --- Complete RPMI-1640 medium / Chapter 5. --- Trypsin-ethylenediaminetetraacetate solution / Chapter 6. --- Phosphate buffered saline / Chapter 7. --- Cycloheximide / Chapter 8. --- Actinomycin D / Chapter 9. --- Trypan blue stain / Chapter 10. --- Neutral red stain / Chapter 11. --- Recombinant human tumour necrosis factor / Chapter 12. --- Cell culture plates and flasks / Chapter 2.3 --- GROWTH CHARACTERISTIC --- p.22 / Chapter 1. --- Tritiated Thymidine / Chapter 2. --- Tritiated Leucine / Chapter 3. --- Trichloroacetic acid / Chapter 4. --- Scintillation cocktail / Chapter 2.4 --- "RESPONSE TOWARDS ANTICANCER DRUGS, CYTOTOXIC AGENTS, AND ENZYME MODULATORS" --- p.23 / Chapter 1. --- N-acetyl-DL-homocysteinethiolactone / Chapter 2. --- Diethyldithiocarbamic acid / Chapter 3. --- Doxorubicin / Chapter 4. --- Acivicin / Chapter 5. --- Ethacrynic acid / Chapter 6. --- "L'Buthionine-[S,R]-sulfoximine" / Chapter 7. --- Hydrogen peroxide / Chapter 8. --- Methotrexate / Chapter 9. --- Menadione / Chapter 2.5 --- CULTURE OF BACTERIAL CELLS --- p.27 / Chapter 1. --- Ampicillin stock solution / Chapter 2. --- Chloramphenicol stock solution / Chapter 3. --- Tetracycline stock solution / Chapter 4. --- Luria-Bertani medium / Chapter 5. --- LB with ampicillin / Chapter 6. --- SOB medium / Chapter 7. --- SOB medium with ampicillin / Chapter 8. --- SOC medium / Chapter 9. --- SB medium / Chapter 10. --- SB medium with ampicillin / Chapter 11. --- Agar plates / Chapter 2.6 --- PREPARATION OF DNA PROBES FROM BACTERIAL CLONES --- p.29 / Chapter 1. --- FlexiPrep Kit / Chapter 2. --- Restriction endonucleases / Chapter 3. --- GeneClean® II Kit / Chapter 4. --- cDNA clones for making DNA probes / Chapter 5. --- TrisHCl EDTA buffer / Chapter 2.7. --- ELECTROPHORESIS OF DNA --- p.31 / Chapter 1. --- EDTA stock solution / Chapter 2. --- Tris acetate EDTA electrophoresis buffer / Chapter 3. --- Tris borate EDTA electrophoresis buffer / Chapter 4. --- Ethidium bromide / Chapter 5. --- DNA molecular size markers / Chapter 6. --- TAE/TBE agarose gel slab / Chapter 2.8 --- CONSTRUCTION OF MURINE TNFR1 PARTIAL cDNA CLONE --- p.33 / Chapter 1. --- Frist strand cDNA synthesis Kit / Chapter 2. --- Murine TNFR1 forward and reverse primers / Chapter 3. --- Polymerase chain reaction reagents / Chapter 4. --- Cloning vector / Chapter 5. --- Modifing enzymes / Chapter 6. --- T7 SequencingTM Kit / Chapter 7. --- Acrylamide/bis gel stock solution / Chapter 8. --- Urea / Chapter 9. --- TEMED and ammonium persulphate / Chapter 10. --- β-Galactosidase colour test reagents / Chapter 11. --- TFB solution / Chapter 12. --- DnD solution / Chapter 2.9. --- RADIOLABELLING OF DNA PROBES --- p.35 / Chapter 1. --- Oligolabelling kit / Chapter 2. --- Redivue [α-32P] dCTP / Chapter 3. --- PUSH column / Chapter 2.10 --- EXTRACTION OF TOTAL RNA FROM CELL LINES --- p.36 / Chapter 1. --- N-Lauroylsarcosine / Chapter 2. --- 2M sodium acetate (pH48) / Chapter 3. --- Phenol / Chapter 4. --- Isopropanol / Chapter 5. --- Ethanol / Chapter 6. --- Extraction buffer / Chapter 7. --- Chloroform / Chapter 8. --- Isoamyl alcohol / Chapter 2.11 --- HYBRIDIZATION AND NORTHERN ANALYSIS --- p.37 / Chapter 1. --- 20XSSC / Chapter 2. --- 5X formaldehyde running buffer / Chapter 3. --- RNA sample buffer / Chapter 4. --- 10X RNA loading buffer / Chapter 5. --- Formaldehyde slab gel / Chapter 6. --- Hybond®-N membrane / Chapter 7. --- Immobilon®-N membrane / Chapter 8. --- Salmon sperm DNA / Chapter 9. --- Sodium dodecyl sulphate / Chapter 10. --- Dextran sulphate / Chapter 11. --- Kodak Biomax MR and X-OMAT films and developing kits / Chapter 2.12 --- APPARATI USED --- p.39 / Chapter CHAPTER 3 --- METHODS / Chapter 3.1 --- ISOLATION AND MAINTENANCE OF TNF RESISTANT L929 CELLS --- p.40 / Chapter 3.1.1 --- Culture of L929 cells / Chapter 3.1.2 --- Trypan blue exclusion test / Chapter 3.1.3 --- Isolation of TNF-resistant variants of L929 / Chapter 3.1.4 --- Verification of the TNF-resistant trait of rL929 / Chapter 3.1.5 --- Neutral red uptake assay / Chapter 3.2 --- COMPARING L929 AND rL929 CELLS IN TERMS OF GROWTH CHARACTERISTICS --- p.43 / Chapter 3.2.1 --- Doubling time / Chapter 3.2.2 --- Rate of protein synthesis / Chapter 3.2.3 --- Rate of DNA synthesis / Chapter 3.3 --- COMPARING L929 AND rL929 CELLS IN TERMS OF RESPONSE TOWARDS DIFFERENT ENZYME INHIBITORS AND CYTOTOXIC AGENTS --- p.44 / Chapter 3.3.1 --- TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.2 --- Effects of inhibitors of gene transcription and protein synthesis on TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.3 --- Cytotoxic effect of hydrogen peroxide and menadione on L929 and rL929 cells --- p.44 / Chapter 3.3.4 --- TNF cytotoxicity on L929 and rL929 cells: effect of N-acetyl homocysteine thiolatone --- p.45 / Chapter 3.3.4.1 --- The tolerant limit of AHCT / Chapter 3.3.4.2 --- Effect of AHCT on TNF cytotoxicity / Chapter 3.3.5 --- TNF cytotoxicity on L929 and rL929 cells: effect of diethyldithiocarbamate --- p.46 / Chapter 3.3.5.1 --- The tolerant limit of DEDTC / Chapter 3.3.5.2 --- Effect of DEDTC on TNF cytotoxicity / Chapter 3.3.6 --- TNF cytotoxicity on L929 and rL929 cells: effect of buthionice sulfoximine --- p.47 / Chapter 3.3.6.1 --- The tolerant limit of BSO / Chapter 3.3.6.2 --- Effect of BSO on TNF cytotoxicity / Chapter 3.3.7 --- TNF cytotoxicity on L929 and rL929 cells: effect of Acivicin --- p.47 / Chapter 3.3.7.1 --- The tolerant limit of acivicin / Chapter 3.3.7.2 --- Effect of acivicin on TNF cytotoxicity / Chapter 3.3.8 --- TNF cytotoxicity on L929 and rL929 cells: effect of ethacrynic acid --- p.48 / Chapter 3.3.8.1 --- The tolerant limit of ethacrynic acid / Chapter 3.3.8.2 --- Effect of ethacrynic acid on TNF cytotoxicity / Chapter 3.3.9 --- Cytotoxic effect of doxorubicin on L929 and rL929 cells --- p.49 / Chapter 3.3.10 --- TNF cytotoxicity of L929 cells: effect of N-acetyl cysteine --- p.49 / Chapter 3.3.11 --- Cytotoxic effect of methotrexate on L929 and rL929 cells --- p.50 / Chapter 3.3.12 --- Cytotoxic effect of hyperthermia on L929 and rL929 cells --- p.50 / Chapter 3.4 --- NORTHERN ANALYSIS AND HYBRIDIZATION --- p.51 / Chapter 3.4.1. --- Preparing RNA blots --- p.51 / Chapter 3.4.1.1 --- Extraction of total RNA from cells / Chapter 3.4.1.2 --- Making equal loading of RNA samples in formaldehyde gel electrophoresis / Chapter 3.4.1.3 --- Northern blotting of RNA / Chapter 3.4.2. --- Preparation of cDNA probes --- p.53 / Chapter 3.4.2.1 --- Preparing plasmids from A TCC clones / Chapter 3.4.2.2 --- Preparing TNFR1 probe from first-strand cDNA of L929 cells / Chapter 1. --- Construction of recombinant clone from the PCR product of TNFRl fragment / Chapter 2. --- Transforming the recombinant vector into JM109 host / Chapter 3. --- Sequencing of PCR product for identity confirmation / Chapter 3.4.2.3 --- Preparing DNA inserts from plasmids / Chapter 3.4.3 --- Radiolabelling of cDNA probes --- p.56 / Chapter 3.4.4 --- Hybridization of radioactive probes to RNA blots --- p.57 / Chapter CHAPTER 4 --- RESULTS AND DISCUSSIONS / Chapter 4.1 --- ISOLATION OF TNF-RESISTANT VARIANTS OF L929 CELLS --- p.58 / Chapter 4.1.1 --- Single cell subcloning of TNF-resistant L929 variants / Chapter 4.1.2 --- Growth rates of L929 and rL929 cells / Chapter 4.1.3 --- Rate of protein synthesis in L929 and rL929 cells / Chapter 4.1.4 --- Rate of DNA synthesis in L929and rL929 cells / Chapter 4.2 --- EFFECT OF INHIBITORS OF GENE TRANSCRIPTION AND PROTEIN SYNTHESIS ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.67 / Chapter 4.3 --- RESPONSE OF L929 AND rL929 CELLS TOWARDS VARIOUS CYTOTOXIC AGENTS --- p.70 / Chapter 4.3.1 --- "Response towards methotrexate, an anti-metabolite used in cancer treatment" / Chapter 4.3.2 --- "Response towards doxorubicin, an chemotherapeutic agent used in cancer treatment" / Chapter 4.3.3 --- "Response towards menadione, a cytotoxic agent known to generate free radicals inside cells" / Chapter 4.3.4 --- Response towards hydrogen peroxide: a highly oxidative agent / Chapter 4.3.5 --- "Response towards hyperthermia, a treatment known to exert oxidative stress on cells" / Chapter 4.4 --- EFFECTS OF MODULATORS OF CYTOSOLIC SUPEROXIDE DISMUTASE ON TNF CYTOTOXICITY ON L929 and rL929 CELLS --- p.77 / Chapter 4.5 --- EFFECT OF MODULATORS OF GLUTATHIONE METABOLISM ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.82 / Chapter 4.5.1 --- "Effects of L-buthionine [S,R] sulfoximine, an inhibitor of glutathione synthesis" --- p.82 / Chapter 4.5.2 --- "Effect of N-acetyl cysteine, a cysteine derivative" --- p.84 / Chapter 4.5.3 --- "Effects of acivicin , an inhibitor of GSH reuptake and recycle" --- p.85 / Chapter 4.5.4 --- "Effect of ethacrynic acid, an inhibitor of glutathione S- transferase" --- p.87 / Chapter 4.6 --- GENE EXPRESSION IN L929 AND rL929 CELLS IN THE COURSE OF TNF CHALLENGE --- p.89 / Chapter 4.6.1 --- Isolation of total RNA from L929 and rL929 cells --- p.89 / Chapter 4.6.2 --- Preparation of DNA probes for hybridization --- p.89 / Chapter 4.6.3 --- Hybridization of specific probes on RNA blots --- p.90 / Chapter 4.6.3.1 --- Expression of heat shock protein --- p.70 / Chapter 4.6.3.2 --- Expression of the p55 TNF receptor / Chapter 4.6.3.3 --- Expression of glutathione reductase / Chapter 4.6.3.4 --- Expression of glutathione S-transferase pi / Chapter 4.7 --- DISCUSSIONS OF THE EXPERIMENTAL RESULTS --- p.97 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.104 / APPENDIX / Generation of the TNF receptor 1 cDNA probe --- p.106 / REFERENCES --- p.108
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_320670 |
| Date | January 1995 |
| Contributors | Kwan, Leo., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, 116 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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