Despite the high mortality rate of ovarian cancer, there are few selective biomarkers that detect its progression and none have become successful targets for therapy. A complex microenvironment that promotes angiogenesis, reduces immune responses and alters the integrity of the surrounding matrix is involved through the biology of ovarian cancer. Previous studies done by our lab and collaborators indicated that extracellular threonyl-tRNA synthetase (TARS) is a pro-angiogenic mediator of the ovarian tumor microenvironment, which is secreted in response to inflammatory signals, and actively promotes angiogenesis. In order to better understand the mechanisms underlying the angiogenic effects of TARS in ovarian cancer, it is essential to identify whether it directly affects ovarian tumor growth and invasion. Preliminary evidence indicated that TARS is secreted from ovarian cancer cells in response to TNF-α and TARS exhibits extracellular angiogenic activity. In previous studies, TARS was shown to significantly increase migration of HUVECs in a transwell assay to an extent that was similar to VEGF. The purpose of this project was to establish a role for TARS in tumor progression and its potential as a diagnostic marker using an animal model of ovarian cancer. The hypothesis tested is that TARS plays a key role in the angiogenic and invasive potential of ovarian cancer, and TARS inhibition will reduce the angiogenic effect of tumor cells which is reflected by measurement of intratumor microvessel density (MVD). The study tested the effect of BC194-mediated TARS inhibition on the development of ovarian tumors in ID8 mouse model. We found a positive correlation between TARS expression and ovarian cancer progression, and TARS inhibition with BC194 reduce the progression of ovarian cancer. These data suggest that TARS has an important role in the tumor microenvironment and that TARS inhibition should be further investigated as a therapy for ovarian and other angiogenic cancers.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1997. / Includes bibliographical references. Also available on the Internet.

Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R. rhodochrous DAP 96622 cells was around 50 units/min·mg-cdw which was the highest of all inducers tested. When induced with Co only, the nitrile hydratase activity of R. rhodochrous DAP 96622 was around 20 units/min·mg-cdw, and the nitrile hydratase activity of R. rhodochrous DAP 96622 uninduced was the same as the nitrile hydratase activity of uninduced R. rhodochrous DAP 96253. When Co/urea induced R. rhodochrous DAP 96253 cell lysate was examined on gradient SDS-PAGE and analyzed by Image Quant TL, the nitrile hydratase bands (both α and β subunits) accounted for more than 55% of the total cytosolic proteins. Whereas in Co/urea induced R. rhodochrous DAP 96622, the nitrile hydratase bands accounted for around 25% of the total cytosolic proteins. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry results, amidase in R. rhodochrous DAP 96253 was approximately 38 kDa from the nitrilase/cyanide hydratase family and amidase in R. rhodochrous DAP 96622 was 55 kDa from the amidase signature family. In addition, the nitrile hydratase regulation system in both R. rhodochrous DAP 96253 and DAP 96622 strains are different. Moreover, the nitrile hydratase regulation system in R. rhodochrous DAP 96253 is different from R. rhodochrous J1. Purified nitrile hydratase from R. rhodochrous DAP 96253 may form a protein complex with glutamine synthetase, resulting in a nitrile hydratase activity of approximately 1500 units/mg-proteins, and nitrile hydratase from R. rhodochrous DAP 96622 is not a protein complex and results in a nitrile hydratase activity of 950 units/mg-proteins.

Glutamine synthetase (GS; EC 6.3.1.2; L-glutamate ammonialigase) catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine. Due to its key role in nitrogen metabolism, including nucleotide, amino acid and urea biosynthesis, the enzyme has been ascribed an extraordinarily long evolutionary history. Thus, GS has been used as a molecular clock to establish phylogenetic relationship between different species. Through the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) system, we obtained the complete cDNA of GS from tilapia cDNA liberary. Furthermore, the results of the alignment of tilapia GS sequence with that of other species indicated a close relationship between tilapia GS and other fishes. We also found that there is 79% homology between mammal and tilapia within the open read frame (ORF) of GS. However, sequence analysis by computer software revealed the fact that the size (0.5 kb) of GS 3¡¦untranslated region (3¡¦-UTR) of tilapia GS is different from that of mammals. Moreover, there is the complete distinct sequence of the 3¡¦-UTR of tilapia GS from that of mammals. The 3'-UTR of many eukaryotic mRNAs has been implicated in the control of mRNA stability, processing, polyadenylation, and translational regulation. Accordingly, to comprehend the role of 3¡¦-UTR in GS phylogenesis, we examine whether the 3'-UTR of tilapia GS is involved in the regulation of GS expression in mammals. We first generated the construct using pEGFP-N2 carrying the ORF (1.1kb) of tilapia GS gene (ORF-GFP) or the full length (1.6kb) of tilapia GS gene (Full-GFP). Transient or stable transfection of C6 gliomal cells with ORF-GFP indicated that GS mRNA and protein was expressed. When C6 cells were stably transfected with Full-GFP, the expression of GS mRNA, but not its protein, was found. Adenine/uridine-rich sequence elements (AREs) of the 3¡¦-UTR have been known to regulate mRNA stability of certain chemokines. Four AREs are also found in the 3¡¦-UTR of tilapia GS. We further generated the constructs with tilapia ORF-GFP and its 3¡¦-UTR containing 1-4 AREs (A1-GFP, A2-GFP, A3-GFP and A4-GFP). Stable transfection of C6 cells with the different constructs indicated that tilapia GS mRNA is normally transcripted, while there was no expression of GS proteins in stable transfectants. The findings suggest tilapia GS protein expression in mammals by its 3¡¦-UTR and unidentified evolutionary role of the 3¡¦-UTR region of GS.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 180-187).

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 81-94. Also available online.

Thesis (Master's Degree (Medical Technology)-- Cape Technikon, Cape Town, 1997 / There is, at present, no recognised diagnostic biochemical marker of Alzheimer's Disease (AD). Recently, Gunnerson and Haley, (1992), reported that the presence of glutamine synthetase (GS) in cerebrospinal fluid (CSF) samples showed a 97% correlation with patients diagnosed as having AD. GS was detected by photolabelling with [y32P]2-azido-ATP or [y32P]8azido- ATP and visualisation following sodium dodecyl sulphate polyacrylamide gel electrophoresis (SOS-PAGE) and autoradiography. This study set out to reproduce Gunnerson and Haley's methodology for labelling sheep GS in CSF using [y32P]8-azidoATP, to develop this assay or possibly another, using a fluorescent probe of ATP binding sites, into a robust procedure suitable for a routine diagnostic laboratory, and finally to assess whether the presence of GS in CSF is indeed a marker of AD.

Methylglyoxal synthetase, which catalyzes the formation of methylglyoxal and inorganic phosphate from dihydroxyacetone phosphate, was found in extracts of Proteus vulgaris. An efficient purification procedure utilizing ion exchange column chromatography and isoelectric focusing has been developed. Homogeneity of the enzyme preparation was confirmed by polyacrylamide gel electrophoresis and rechromatography.Two components of methylglyoxal synthetase were obtained upon isoelectric focusing. A comparison of the chemical and physical properties of the two components was carried out. The enzyme is a dimer. In the presence of inorganic phosphate, the hyperbolic saturation kinetics with dihydroxyacetone phosphate are shifted to sigmoidal.

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, and aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.

The composition, size, and peptide structure of a fatty acid synthetase aggregate from etiolated Euglena gracilis was studied. The fatty acid synthetase was a lipoprotein containing about 40% lipid. Low-angle laser light scattering of the native fatty acid synthetase yielded a molecular weight of 6 · 106 up to concentrations of about 30 μg fatty acid synthetase/ml; at higher concentrations, the molecular weight increased to 11 · 106. Viscometry of the synthetase solutions yielded results that suggested that the asymmetric fatty acid synthetase aggregate formed a 'dimer' at concentrations above 30 μg fatty acid synthetase/ml by side-to-side interaction. The peptide structure of the fatty acid synthetase prepared in the presence of a variety of proteinase inhibitors included at least six peptides of Mr 150000 or less. More than 68% of the protein was in peptides of less than Mr 150000. N-terminal amino acid analysis gave eight different residues all present in integral amounts, seven at about 11% and one at 24% of the total α-N-dansyl amino acids. The Euglena-aggregated fatty acid synthetase appears to be a very large true multienzyme complex.