ABSTRACT Cassava is a known source of linamarin, but difficulties associated with its isolation have prevented it from being exploited as a source. A batch adsorption process using activated carbon at the appropriate contact time proved successful in its isolation with ultrafiltration playing a pivotal role in the purification process. Result revealed that optimum purification was obtained with increasing amount of crude cassava extract (CCE) purified. 60g of CCE took 32 mins, 80 g, 34 mins while 100 g took 36 mins of contact time, where 1.7 g, 2.0 g and 2.5 g of purified product were obtained, respectively. The purification process in batch mode was also carried out at different temperatures ranging from 25 to 65oC. Results showed that purification increases with increase in temperature. In a bid to ascertain the moles of linamarin adsorbed per pore volume of activated carbon used, the composite isotherm was found to represent the measured adsorption data quite well. The adsorption of linamarin was used to study the goodness of fit criteria (R2) for the entire process. Results showed that R2 value was best with decreasing amount of CCE purified (R2=1 for 60 g) at the temperature of 45oC. Compound elucidation of purified product by Picrate paper test, IR and 1HNMR confirmed the structure of linamarin. Cytotoxic effects of linamarin on MCF-7, HT-29, and HL-60 cells were determined using the 3 - (4, 5 – dimethylthiazol-2-yl) – 2, 5 – diphenyltetrazolium bromide (MTT) assay. Cytotoxic effects were significantly increased in the presence of linamarase, which catalysed the hydrolysis of linamarin to hydrogen cyanide. A 10–fold decrease in the IC50 values obtained for linamarin or crude extract in the presence of linamarase was determined for HL-60 cells. This study thus describes a method for the isolation and purification of linamarin from cassava, as well as the potential of this compound as an anticancer agent.

Tam Wai-Kwan Karen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 109-128). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiv / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature Review --- p.2 / Chapter 2.1 --- Introduction of Cordyceps --- p.2 / Chapter 2.2 --- Pharmacological functions of Cordyceps --- p.3 / Chapter 2.2.1 --- Functions in respiratory system --- p.3 / Chapter 2.2.2 --- Functions in renal system --- p.7 / Chapter 2.2.3 --- Functions in hepatic system --- p.8 / Chapter 2.2.4 --- Functions in cardiovascular system --- p.9 / Chapter 2.2.5 --- Functions in endocrine and steroid system --- p.10 / Chapter 2.2.6 --- Functions in the immune system --- p.11 / Chapter 2.2.7 --- Functions in nervous system --- p.15 / Chapter 2.2.8 --- Controls in glucose metabolism --- p.15 / Chapter 2.2.9 --- Anti-oxidation activity --- p.16 / Chapter 2.2.10 --- Anti-tumor activity --- p.18 / Chapter 2.3 --- Active ingredients of Cordyceps and their related biological activities --- p.20 / Chapter 2.3.1 --- Polysaccharides --- p.20 / Chapter 2.3.2 --- Nucleosides --- p.21 / Chapter 2.3.2.1 --- Adenosine --- p.21 / Chapter 2.3.2.2 --- Cordycepin --- p.24 / Chapter 2.4 --- Proteomic tools in studies of the change in protein expression --- p.25 / Chapter 2.4.1 --- Two-dimensional electrophoresis --- p.27 / Chapter 2.4.2 --- Mass Spectrometry --- p.28 / Chapter 3. --- Methods and Materials --- p.30 / Chapter 3.1 --- Cell lines and culture conditions --- p.30 / Chapter 3.2 --- Trypan blue exclusion method --- p.30 / Chapter 3.3 --- Cell counting --- p.31 / Chapter 3.4 --- Anti-proliferation assay --- p.31 / Chapter 3.5 --- Anti-proliferation assay of normal cell line --- p.32 / Chapter 3.6 --- Determination of ic50 --- p.33 / Chapter 3.7 --- Sample preparation for proteins studies --- p.33 / Chapter 3.8 --- Protein quantitation --- p.34 / Chapter 3.9 --- Gel electrophoresis --- p.36 / Chapter 3.10 --- Image analysis --- p.37 / Chapter 3.11 --- In-gel digestion and MALDI-ToF MS --- p.37 / Chapter 3.12 --- Statistical Analysis --- p.39 / Chapter 3.13 --- Chemicals --- p.39 / Chapter 4. --- Results --- p.41 / Chapter 4.1 --- MTT assay --- p.41 / Chapter 4.1.1 --- The anti-proliferating activity of adenosine against cancer cell lines (HepG2 and SV7tert) and normal cell line (Hs68) --- p.41 / Chapter 4.1.2 --- The anti-proliferating activity of cordycepin against cancer cell lines (HepG2 and SV7tert) and normal cell line (Hs68) --- p.42 / Chapter 4.1.3 --- The anti-proliferation effects of adenosine and cordycepin --- p.42 / Chapter 4.2 --- Changes in protein expression --- p.50 / Chapter 4.2.1 --- "Corresponding drug treatment of cell lines (HepG2, SV7tert and Hs68)" --- p.50 / Chapter 4.2.2 --- "Comparison of protein profiles from cells (HepG2, SV7tert or Hs68) under the normal and drug treated (with either adenosine or cordycepin) conditions" --- p.51 / Chapter 4.2.2.1 --- HepG2 study --- p.51 / Chapter 4.2.2.2 --- SV7tert study --- p.52 / Chapter 4.2.2.3 --- Hs68 study --- p.52 / Chapter 4.2.3 --- Protein identification --- p.53 / Chapter 4.2.3.1 --- HepG2 cell line --- p.53 / Chapter 4.2.3.2 --- HepG2-changes in protein expressions after adenosine treatment --- p.54 / Chapter 4.2.3.3 --- HepG2-changes in protein expressions after cordycepin treatment --- p.54 / Chapter 4.2.3.4 --- SV7tert cell line --- p.54 / Chapter 4.2.3.5 --- SV7tert-changes in protein expressions after cordycepin treatment --- p.55 / Chapter 4.2.3.6 --- Hs68 cell line --- p.55 / Chapter 4.2.3.7 --- Hs68-changes in protein expressions after cordycepin treatment --- p.56 / Chapter 5. --- Discussion --- p.89 / Chapter 5.1 --- anti-proliferation assays --- p.89 / Chapter 5.2 --- changes in protein expression: --- p.90 / Chapter 5.2.1 --- Protein alterations in HepG2 --- p.91 / Chapter 5.2.1.1 --- Changes in protein expression (membrane protein and transport: Trimethyllysine hydroxylase) --- p.91 / Chapter 5.2.1.2 --- Changes in protein expression (protein synthesis and folding: carboxypeptidase E) --- p.92 / Chapter 5.2.1.3 --- Changes in protein expression (membrane proteins and transport: calumenin and electron transfer flavoproteins) --- p.93 / Chapter 5.2.2 --- Protein alterations in SV7tert --- p.94 / Chapter 5.2.2.1 --- Changes in protein expression (protein synthesis and folding: BiP(GRP78)) --- p.94 / Chapter 5.2.2.2 --- Changes in protein expression (cell defense and tolerance: Hsp60 (chaperonin); TANK binding kinase-1) --- p.96 / Chapter 5.2.2.3 --- Changes in protein expression (metabolism: prolyl 4-hydroxylase; aldolase A; glyceraldehyde-3-phosphate dehydrogenase) --- p.97 / Chapter 5.2.2.4 --- Changes in protein expression (cell growth and division: βII tubulin; HnRNP Al) --- p.100 / Chapter 5.2.3 --- Protein alterations in Hs68 --- p.101 / Chapter 5.2.3.1 --- Changes in protein expression (metabolism: triosephosphate isomerse 1) --- p.101 / Chapter 6. --- Discussion --- p.103 / Chapter 6.1 --- The antiproliferating activities of adenosine and cordycepin --- p.103 / Chapter 6.2 --- "Effects of adenosine and cordycepin on the changes in protein expressions in HepG2, SV7tert and Hs68" --- p.104 / Chapter 6.3 --- Problems and improvements in two-dimensional gel electrophoresis --- p.105 / Chapter 7. --- Conclusion and future prospectives --- p.107 / References --- p.109

No description available.

As fish cell cultures continue to be explored as alternatives to whole fish for evaluating the toxicity of environment chemicals, technical issues have emerged that influence results and thus need to be understood and standardized. These include carrier solvents, dosing protocols, exposure vessel, exposure media, viability endpoints, and cell lines. Some of these factors have been explored in this thesis for eight reactive contaminants exhibiting varied physicochemical properties using the rainbow trout cell lines RTgill-W1 and RTL-W1. Sodium dodecyl sulphate (SDS) was used as a reference (control) chemical. Cell viability was evaluated with alamar Blue, carboxyfluoroscein diacetate acetoxymethyl ester and neutral red as measures respectively of metabolic activity, plasma membrane integrity, and lysosomal function. Experimental in vitro EC50 values were compared to 1) pre-existing in vivo LC50s from the fathead minnow database and 2) pre-existing in vitro EC50s from the Halle database. Results point to good in vitro/in vivo correlations for menadione, dichlorophene, hexachlorophene, and acrolein. Poor correlations were observed for allyl alcohol, 4-fluoroaniline, acetaldehyde, and 2,3-dimethyl-1,3-butadiene due to a combination of solubility and volatility problems. Overall, the results suggest that the impact of different technical approaches on the evaluation of acute toxicity in vitro depends very much on the chemical class being investigated and less on the characteristics of the cell line. The in vitro cytotoxicity of reactive chemicals is challenging due to the nature of the chemicals’ physicochemical properties. Further improving the in vitro toxicity of reactive chemicals is a prerequisite for the ultimate goal of using fish cell cultures as acceptable, standard alternatives to the use of fish acute lethality assays.

As fish cell cultures continue to be explored as alternatives to whole fish for evaluating the toxicity of environment chemicals, technical issues have emerged that influence results and thus need to be understood and standardized. These include carrier solvents, dosing protocols, exposure vessel, exposure media, viability endpoints, and cell lines. Some of these factors have been explored in this thesis for eight reactive contaminants exhibiting varied physicochemical properties using the rainbow trout cell lines RTgill-W1 and RTL-W1. Sodium dodecyl sulphate (SDS) was used as a reference (control) chemical. Cell viability was evaluated with alamar Blue, carboxyfluoroscein diacetate acetoxymethyl ester and neutral red as measures respectively of metabolic activity, plasma membrane integrity, and lysosomal function. Experimental in vitro EC50 values were compared to 1) pre-existing in vivo LC50s from the fathead minnow database and 2) pre-existing in vitro EC50s from the Halle database. Results point to good in vitro/in vivo correlations for menadione, dichlorophene, hexachlorophene, and acrolein. Poor correlations were observed for allyl alcohol, 4-fluoroaniline, acetaldehyde, and 2,3-dimethyl-1,3-butadiene due to a combination of solubility and volatility problems. Overall, the results suggest that the impact of different technical approaches on the evaluation of acute toxicity in vitro depends very much on the chemical class being investigated and less on the characteristics of the cell line. The in vitro cytotoxicity of reactive chemicals is challenging due to the nature of the chemicals’ physicochemical properties. Further improving the in vitro toxicity of reactive chemicals is a prerequisite for the ultimate goal of using fish cell cultures as acceptable, standard alternatives to the use of fish acute lethality assays.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

MTHFD is a folate-dependent trifunctional protein comprised of three activities: N$ sp5$,N$ sp{10}$-methylenetetrahydrofolate dehydrogenase, N$ sp5$,N$ sp{10}$-methenyltetrahydrofolate cyclohydrolase and N$ sp{10}$-formyltetrahydrofolate synthetase. The enzymes catalyse the sequential interconversion of tetrahydrofolate derivatives required for purine, methionine and thymidylate synthesis. A Chinese hamster ovary cell line, reported to have reduced cyclohydrolase activity, was studied to characterize the nature of its mutation. / Enzymatic assays showed reduced activities of all three enzymes. Immunoblotting and immunoprecipitation of radiolabelled cell extracts indicated that the gene product was greatly reduced or absent in the mutant. Southern analysis showed no differences between normal and mutant cells, indicating that the defect was not due to a major gene rearrangement. RNA analysis, by Northern blotting and by RNA amplification using the polymerase chain reaction, showed that a mRNA for MTHFD of normal size was present in mutant cells. These results suggest that the mutation is post-transcriptional and that it disrupts the synthesis of MTHFD.

Clonal cell lines were isolated from mammary gland tissue epithelial cell cultures of lactating cows. Early passage clonal bovine mammary epithelial cells (clone LMH17) gave rise to several established cell lines (MAC-T lines) after being cotransfected with plasmids containing the temperature sensitive mutant SV40 large T antigen gene (pBAPSV40TtsA58) and the bacterial phosphotransferase gene (pSV2-neo). Unlike other cell types which were transformed after being transfected with SV40, MAC-T cells maintained many characteristics of non-transformed cells: MAC-T cells were serum and anchorage dependent, showed contact inhibition, and were not tumorigenic in immunodeficient mice. However, Southern transfer analysis revealed an integrated SV40 gene and cells showed no senescence after 50 passages. These cells are morphologically indistinguishable from parental LMH17 cells and retain the typical morphology of mammary epithelial cells. Positive cytokeratin immunostaining and the absence of vimentin staining indicated that these cells were epithelial in origin. / MAC-T cells grew rapidly on plastic substratum with a doubling time of approximately 17 hours and became differentiated when grown on floating collagen gels in the presence of prolactin. The differentiated phenotype was characterized to include (1) the ability to form secretory domes with a lumen from a pavement of columnar cells; (2) increased casein mRNA abundance; (3) increased alpha S and beta casein secretion; (4) increased number and size of casein secretory vesicles; and (5) increased lactose synthesis and secretion.

While extirpative surgery is curative for localized clear cell renal cell carcinoma (ccRCC), many patients develop recurrences or present with metastatic disease. Several aspects of ccRCC biology have been investigated, but these have been done in cell lines, which are known to poorly represent the tumour. Since cell lines are amenable to a wide array of experimental testing, the studies presented here demonstrate a novel method to generate ccRCC cell lines from primary tumours, which increases the rate of primary tumour cell line generation four fold. Additionally, ccRCC cells do not grow in serum-free media, which has been shown to be beneficial in other cancers. Therefore, we interrogated the effect of exogenous growth factors to optimize our serum-free media growth conditions, among which TGFb1 appeared to elicit the largest mitogenic effect. Once optimized, these findings will provide a valuable tool for understanding ccRCC tumour cell biology and identifying therapeutic targets.

While extirpative surgery is curative for localized clear cell renal cell carcinoma (ccRCC), many patients develop recurrences or present with metastatic disease. Several aspects of ccRCC biology have been investigated, but these have been done in cell lines, which are known to poorly represent the tumour. Since cell lines are amenable to a wide array of experimental testing, the studies presented here demonstrate a novel method to generate ccRCC cell lines from primary tumours, which increases the rate of primary tumour cell line generation four fold. Additionally, ccRCC cells do not grow in serum-free media, which has been shown to be beneficial in other cancers. Therefore, we interrogated the effect of exogenous growth factors to optimize our serum-free media growth conditions, among which TGFb1 appeared to elicit the largest mitogenic effect. Once optimized, these findings will provide a valuable tool for understanding ccRCC tumour cell biology and identifying therapeutic targets.