Several model systems were investigated to elucidate the mechanisms by which rat liver phosphatidylcholine synthesis is controlled. CTP: phosphocholine cytidylyltransferase was clearly the key regulatory enzyme for phosphatidylcholine formation from choline. This ambiquitous enzyme was detected in both the cytosolic and microsomal fractions of rat liver, although the majority of the cytidylyltransferase occurred in the soluble fraction. The distribution of cytidylyltransferase between these fractions was altered when the rate of phosphatidylcholine synthesis was perturbed. Translocation of cytidylyltransferase was observed in rat liver during early development, with starvation and during a diurnal rhythm. A redistribution of cytidylyltransferase was also detected in isolated hepatocytes which were treated with glucagon, cAMP analogues or fatty acids bound to albumin. The rate of phosphatidylcholine synthesis was found to reflect the amount of microsomal cytidylyltransferase activity. The inhibition of phosphatidylcholine synthesis by glucagon or cAMP analogues was likely due to phosphorylation and inhibition of the cytidylyltransferase. Several lines of evidence indicated that the cytidylyltransferase in fresh rat liver cytosol was probably phosphorylated and activated upon dephosphorylation by endogenous phosphoprotein phosphatases or alkaline phosphatase from hog intestine. Although the phosphorylation of cytidylyltransferase was apparently kinetically "silent", dephosphorylation resulted in an increased affinity of the enzyme for membranes. Fatty acids stimulated de novo phosphatidylcholine synthesis by acceleration of the cytidylyl-transferase-catalyzed reaction. Fatty acids and their CoA derivatives were shown to stimulate the cytosolic cytidylyltransferase activity. However, these compounds failed to activate partially purified cytidylyltransferase appreciably. Apparently, fatty acids, like dephosphorylation, enhanced the tenacity of cytidylyltransferase for membranes. Upon binding to membranes, cytidylyltransferase activity could be elevated up to 45-fold, and the affinity of the enzyme for the substrate, CTP, was increased 20-fold. The influence of glucagon, cAMP analogues and fatty acids on the synthesis of phosphatidylcholine by successive N-methylation was also examined in isolated rat hepatocytes. Glucagon and cAMP analogues inhibited the methylation pathway in these cells, but the activity of microsomal phosphatidylethanolamine methyltransferase was elevated. Fatty acids also reduced the formation of phosphatidylcholine from phosphatidylethanolamine. Fatty acids and their CoA derivatives directly inhibited the phosphatidylethanolamine methyltransferase in rat liver microsomes. The coordinate control of hepatic phosphatidylcholine synthesis by cAMP and fatty acids may be important during starvation when the intracellular levels of these compounds are increased. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This study was performed to determine the rates of in vitro cholesterol synthesis, as measured by ³H incorporation into cholesterol, in gluteal white adipose tissue and the intestine of infant rats during the early postnatal period. The reason for studying these parameters was to hopefully further elucidate the cause of the differences in blood cholesterol levels between rats raised in different litter sizes. Rats were raised in small (4/dam), medium (8/dam) and large (14/dam) litters. The rate of cholesterol synthesis in the proximal and distal region of the small intestine decreased from birth to a low 14 days later and then increased again by day 21. The rate of the decreases in cholesterol synthesis, from birth to the low on day 14, varied amongst the three litter sizes; the rate of synthesis in both regions of the intestine on day 7 was lowest in the larger litter, and was not significantly different from the values seen on day 14. Previous work has shown that plasma cholesterol levels were low in rats from large litters on day 7, thus in the early postnatal period low rates of intestinal cholesterol synthesis correlate with low plasma cholesterol values. After weaning, on day 21, the rate of synthesis in distal intestine was higher than that of proximal intestine in the medium and small litters, whereas the opposite was found in the larger litter. Cholesterol synthesis in gluteal white adipose tissue remained at a very low rate up to 21 days in the small and medium sized litters. In contrast, the rate of synthesis increased continuously in the large litter to nearly threefold the rate of that in the other two litter sizes on day 21. This study has shown that the rates of cholesterol synthesis in intestinal and adipose tissue can be altered during the early postnatal period by raising the rats in different litter sizes. Also, the pattern of development in the rates of cholesterol synthesis as measured by ³H incorporation into cholesterol are in general agreement with results reported for the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase for this same period in development. / Land and Food Systems, Faculty of / Graduate

A new approach in mapping restriction fragments by means of primed extension was proposed but was found to be unfeasible after studying the extent of T4 polymerase mediated DNA synthesis. The maximum length of DNA replication mediated by T4 polymerase was studied using ØX-174 DNA as template primed by a restriction fragment of the same DNA. Both nucleotide incorporation kinetics and alkaline gel electrophoresis were used to study the products of DNA synthesis. Although the incorporation kinetics suggested that the primer was extended by approximately 100 nucleotides, the electrophoretic mobilities of the products suggested much less extension. The effect of T4 gene 32 protein (unwinding protein) was also studied. This protein was purified by DNA cellulose chromatography to near homogeneity and was shown to be nuclease free. The purified protein stimulated nucleotide incorporation three-fold when added to the usual T4 polymerase reaction mixture. Contrary to the kinetic results, however, the gel mobilities of the products again showed only limited extension of the primer. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The work presented in this thesis is aimed at the total synthesis of 14,21-dehydrosecodine (1). This substance is an indole derivative with reactive substituents at position 2 (an acrylic ester segment) and 3 (a 1,6-dihydropyridine system). The stabilization of the latter involved the generation of chromium carbonyl complexes employing trisacetonitriletricarbonylchromium (0) as the reagent with appropriate synthetic indole derivatives. In order to develop the required methodology for the preparation of the above complexes, the initial experiments employed simple dihydropyridine systems. Thus, when N-methyl-3-ethyl pyridinium iodide (4_1) was treated with NaBH^ in a two-phase system (ether - water), N-methyl-3-ethyl-1,2-dihydropyridine (46_) was obtained. When this compound was treated with the above complexing agent a mixture (ratio 1:2) of (N-methyl-3-ethyl-l,2-dihydropyridine) tricarbonylchromium (0) (4_3) and (N-methyl-3-ethyl-l, 6-dihydropyridine) tricarbonylchromium (0) (4_4) was obtained. Thermal isomerization of this mixture in refluxing cyclo-hexane afforded a 1 : 1 ratio of (4_3) and (4_4) . Liberation of the organic ligand could be achieved by stirring (43) and/or (4_4) with pyridine. The above strategy was applied to the indole intermediate, N-(2-carbomethoxymethyltryptophyl)-3-ethylpyridinium per-chlorate (_36) but only a low yield (2%) of the desired chromium complexes was obtained. These results prompted a change in the original synthetic strategy and a new approach was initiated by other coworkers in this laboratory. Some studies with the novel system (4_6) were conducted as they relate to position of alkylation. It was shown that (46) undergoes reaction with benzyl bromide to afford the 5-substituted derivative. / Science, Faculty of / Chemistry, Department of / Graduate

Experiments with ØW-14-infected, thymidine-requiring mutants of P_. acidovorans strain 29 demonstrated that deoxyuridine but not thymidine was a precursor of thymine in ØW-14 DNA. Deoxyuridine was also a precursor of the a-putrescinylthymine found in ØW-14 DNA. The biosynthesis of a-putrescinylthymine and thymine was mediated by enzyme activities appearing after infection. ØW-14 DNA synthesis and DNA modification was resistant to the antibiotics trimethoprim and 5-fluorodeoxyuridine. This indicated that endogenous thymidine biosynthesis was unlike that observed in the uninfected host or in other biological systems. These observations helped demonstrate that hydroxy-methyluracil-containing nucleotides were precursors of thymine and a-putrescinylthymine-containing nucleotides (Neuhard et al., 1980). The absence of a-putrescinyl thymine and thymine nucleotides in 0W-14-infected cell nucleotide pools suggested that these nucleotides might be synthesized from hydroxymethyluracil at the polynucleotide level. Degradative analysis of nascent ØW-14 DNA demonstrated the presence of hydroxymethyluracil. Enzymatic degradation of pulse-labelled, nascent 0W-14 DNA followed by TLC suggested the presence of three or more novel nucleotides not found in uniformly labelled DNA samples. These observations were consistent with neutral CsCl analysis of pulse-labelled ØW-14 DNA. This DNA contained unusual heavy density components. ØW-14 ts and amber mutants were screened for defects in DNA replication or DNA modification by CsCl gradient and/or degradative analysis. Some DO mutants were identified. In addition, two DNA modification mutants were found. Am 42 made ØW-14 DNA containing lower-than-normal levels of a-putrescinylthymine and increased levels of thymine. Am 37 accumulated intermediates in a-putrescinylthymine biosynthesis. The conditionally lethal nature of the DNA modification lesion was demonstrated. DNA synthesis was adversely affected by this mutation but DNA precursor supplies were not impaired. Two atypical mononucleotides were purified from am 37 DNA. One was identified as hydroxymethyldeoxyuridylate. The second nucleotide was an acid-labile derivative of hydroxymethyldeoxyuridylate. Analysis of [6- ³H]-uracil and ³²PO₄ labelling ratios, chemical and enzymatic degradation and chromatographic analysis of this nucleotide demonstrated that it was the novel compound 5-(hydroxymethyl-0-pyro-phosphoryl)-deoxyuridylate (abbreviated to hmPPdUMP). 5-(hydroxymethyl-O-pyrophosphoryl)-uracil was shown to be a precursor of a-putrescinylthymine by in vitro modification of am 37 DNA with ØW-14 wild-type infected P. acidovorans cell-free extracts. In vitro modification confirmed that a-putrescinylthymine was formed at the polynucleotide level. ØW-14 DNA modification was not necessarily coupled to replication. The presence of hydroxymethyluracil in am 37 DNA agreed with the suggestion that hmPPura was formed by pyrophos-phorylation of hydroxymethyluracil in nascent DNA. HmPPdUMP had chromatographic properties similar to one of the compounds detected in pulse-labelled ØW-14 wild-type DNA. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In Part I, a modified synthesis of radio-labelled secodine (68) and its incorporation into vindoline (7) is described. In a model study, for the synthesis of side-chain labelled 3-ethylpyridine (74), [2-² H]-(3'-pyridyl)-ethane was achieved from the correspondingly labelled 3-acetylpyridine by desulphurization of the intermediate thioketal (93). In a second study, [1- ³H]-(3'-pyridyl)- ethane was synthesized by treating 3-acetylpyridine with sodium borohydride-³H. The resulting alcohol (95) was acetylated, and hydrogenolysis achieved the desired product. The ester alcohol (74) was coupled to [1- ³H]-(3'-pyridyl)-ethane and the resulting pyridinum salt (90) was reduced to the corresponding piperdeine ester (80) in a "one-pot" synthesis. The conversion of (80) to [19-³H]-secodine was achieved by a known procedure. In two experiments, [19-³H, ¹⁴C0₂CH₃]-secodine (68)(³H/¹³C ratios = 3.00 and 1.54) was administered to Catharanthus roseus plants. The vindoline (7) which was isolated was shown to have been biosynthesized from the entire secodine molecule (³H/¹³C = 3.31 and 1.35 respectively). In Part II, a degradation scheme designed to achieve the isolation of the N-methyl group of uleine (1) is described as well as preliminary results from an investigation into the biosynthesis of uleine (1) and olivacine (4). Variously radio-labelled forms of tryptophan (15), anthranilic acid and secodine (18) were administered to Aspidosperma pyricollum root segments and whole plants. The uleine (1) which was isolated was found to be inactive in all experiments. Variously radio-labelled forms of tryptophan (15), anthranilic acid and secodine (18) as well as ¹⁴CH₃-methionine (30) was administered to Aspidosperma australe plants. Uleine (1) and olivacine (4) was isolated. The only incorporation that could be demonstrated was that of ¹⁴CH₃ methionine (30) into uleine (1) to the extent of 0.168% and 0.147%. The isolation of the N-methyl group from (1) showed that it contained 97% and 98% of the activity. In Part III, the attempted synthesis of compounds of the preakuammicine- and stemmadenine-series is described. A new method for the C-18 deoxygenation of curan derivatives using Birch reduction conditions was achieved. Also, a modification of the Oppenauer oxidation of the curenol (36) to achieve improved yields of the aldehyde (37) and nor-fluorocurarine (39) was developed. The introduction of a carbomethoxy group into the C-16 position of the curan aldehyde derivatives (44) and (50) using a base and methylchloroformate was unsuccessful. Also, the introduction of cyanide into position C-16 of the indole alcohol (52) or indole acetate (57) via the corresponding chloroindolenines was unsuccessful. The synthesis of product (60), which is believed to be identical with preakuammicine aldehyde (7), was achieved. This material could not be converted into akuammicine (5) or stemmadenine (4). Only the dehydrated indolenine (72) could be obtained. The ring-opening reaction of the corresponding thioacetal derivative (73) yielded the decarboxylated indole thioacetals (75) and (76). / Science, Faculty of / Chemistry, Department of / Graduate

Anaemia frequently accompanies chronic inflammatory diseases like rheumatoid arthritis and cancer. It is postulated to result primarily from the suppression of erythropoiesis by inflammatory cytokines. A contributing factor could be the inhibition of erythropoietin synthesis which may also be mediated by cytokines. Erythropoietin is the hormone which regulates erythropoiesis. The aims of this project were to investigate whether cytokines can indeed suppress erythropoietin production, and to determine whether the erythropoietin response in experimental models of acute and chronic inflammation was appropriate for the associated anaemia. Macrophage-conditioned medium, interleukin-1β, interleukin-6, tumour necrosis factor-α, and neopterin were assayed for inhibition of erythropoietin synthesis by HepG2 cells in culture. All, except neopterin, effected dose-dependent reductions in the secretion of the hormone. Interleukin-1β and tumour necrosis factor-α down-regulated erythropoietin gene transcription, whereas interleukin-6 inhibited a post-transcriptional process. Rats with acute inflammation developed a mild anaemia which evoked an increase in their serum levels of erythropoietin. The serum erythropoietin levels were optimal, since rats with acute inflammation and severe phenylhydrazine-induced anaemia did not have lower levels of the hormone than controls with a similar degree of anaemia, but without acute inflammation. Erythropoietin is, therefore, not an acute phase reactant. Mice with cancer developed a progressive anaemia which was not due to bone marrow invasion by tumour cells. During the first fourteen days after inoculating them with cancer cells, the mice responded by increasing their serum levels of erythropoietin as the anaemia worsened. The erythropoietin response was appropriate when compared to mice with the same degree of phenylhydrazine-induced anaemia. Erythropoietin levels measured in mice with tumours older than fourteen days were significantly lower than those of control mice with the same degree of experimental anaemia. These animals were very cachectic, suggesting that a blunted erythropoietin response may depend on disease activity.

There is much evidence in the recent literature that peptides may be intermediates in normal protein biosynthesis. This has also been inferred from certain disease states and other conditions under which protein biosynthesis is blocked at some point, e.g. cadmium, amino acid analogues or (in bacteria) antibiotics. The literature covering this concept will be presented. The present studies have been carried out on children, who because they are suffering from chronic protein malnutrition have very much lowered rates of protein synthesis and breakdown. In this unfortunate, but natural experiment, it was hoped that some factor or factors derived from protein synthesis might be found which influenced the synthetic mechanism as a whole. Evidence from the literature has been summarized which concludes that urine, apart from being convenient to collect, is the biological fluid most likely to contain high concentrates of peptides which are released during cellular metabolism.

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