Bibliography: leaves 359-376.

5'-Deoxy-5'-methylthioadenosine (MTA) is derived from s-adenosylmethionine (AdoMet) during the synthesis of the polyamines spermidine and spermine. Methionine can be regenerated from MTA by one of two mechanisms. In mammalian cells and some microorganisms, MTA is degraded to adenine and 5-methylthioribose-1-phosphate (MTR-1-P) via MTA phosphorylase. In certain other microbes, however, MTA is catabolized in two steps; first to adenine and 5-methylthioribose (MTR) via MTA nucleosidase followed by conversion of MTR to MTR-1-P via MTR kinase. This study was to demonstrate the presence of MTA nucleosidase or MTA phosphorylase in both redia containing cercariae and adult Fasciola hepatica Linnaeus, 1758. If MTA nucleosidase was present, it was wanted to determine if MTR kinase was also present. The phosphate-dependent cleaving activity of MTA phosphorylase was demonstrated in the cell-free extracts of adult Fasciola hepatica along with an unidentified MTR metabolizing activity. Redia containing cercariae showed MTA nucleosidase and MTR kinase activity.

Bibliography: leaves 359-376 / 376 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agronomy, 1981

Malaria presents a global health risk that is becoming increasingly difficult to treat due to increased resistance of both the parasite and mosquito to all known drugs. Identification of novel drug targets are therefore essential in the fight against malaria. Polyamines are small flexible polycations that are represented by three basic polyamines. The interaction of polyamines with various macromolecules may lead to stabilisation of DNA, regulation of transcription, replication, and also have an important role in cellular differentiation, proliferation, growth and division. Therefore, disruption of polyamine biosynthesis presents a unique drug target worth exploiting. Polyamine biosynthesis in P. falciparum is regulated by a unique bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) complex, which is unique to P. falciparum and differs completely from human polyamine biosyntehsis. The inhibition of AdoMetDC induces spermidine and subsequent spermine depletion within the parasite that ultimately results in cell cycle arrest. A functional genomics approach was used within this study to identify a global response of the parasite due to the inhibition of AdoMetDC with the irreversible inhibitor, MDL73811. The proteomics approach was optimised for conditions specific to our laboratory with regard to protein extraction, Plasmodial protein quantification, spot detection and finally protein identification by mass spectrometry (MS). This methodology resulted in reliable spot detection and achieved a 95% success rate in MS/MS identification of protein spots. Application of this methodology to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots from the Plasmodial ring and trophozoite stages, which also confirmed stage specific protein production. Various protein isoforms were present which may be of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle. Subsequent application of the 2-DE methodology to the proteome of AdoMetDC inhibited parasites resulted in the identification of 61 unique Plasmodial protein groups that were differentially affected by the inhibition of AdoMetDC in 2 time points. The transcriptome of AdoMetDC inhibited parasites were also investigated at 3 time points. Investigation into the transcriptome revealed the differential regulation of 549 transcripts, which included the differential regulation of polyamine specific transcripts. Inhibition of AdoMetDC provided a unique polyamine specific transcriptomic signature profile that demonstrated unique interactions between AdoMetDC inhibition and folate biosynthesis, redox metabolism and cytoskeleton biogenesis. The results presented provide evidence that the parasite responds to AdoMetDC inhibition by the regulation of the transcriptome and proteome in an attempt to alleviate the effects of AdoMetDC inhibition. Further analyses of the metabolome also provided evidence for the tight regulation of the AdoMet cycle. Overall, this study demonstrated important functional consequences as a result of AdoMetDC inhibition. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

Two experiments were conducted with a total of 1,728 Large White turkeys to determine the relative potencies of four methionine compounds on an equimolar basis. A 28% protein basal diet composed primarily of ground yellow corn, dehulled soybean meal, and meat and bone meal was supplemented with DL-methionine (DL), sodium salt of DL-methionine (MENA), methionine hydroxy analogue calcium salt (MHAC), or methionine hydroxy analogue free acid (MHAA) at the .06, .12, .18, .24, or .3090 level of added methionine. Each of these 20 diets was fed to two pens of poults of each sex (9 birds per pen), and the basal diet was fed to eight pens of each sex from one day to sevenweeks of age in each experiment. From the combined six-week body weight data, MENA was 104.7 (86.1, 123.3), MHAC 101.8 (83.5, 120.1), and MHAA 92.590 (74.9, 110.1) as potent as DL (with 9590 fiducial limits) by the slope ratio procedure. No differences were detectable among the methionine products. The extra one degree of freedom associated with the nonlinear procedure accounted for 4.8 and 10. 7% of the residual variation in Experiments 1 and 2, respectively. Relative potencies were obtained for MENA of 110.3 and 115.6, MHAC of 100.4 and 88.1, and MHAA of 89.7 and 112.390 in Experiments 1 and 2, respectively. Two additional experiments were conducted using a total of 1,680 poults to study the relationships of methionine, choline, and sulfate in practical-type diets. A 6 x 2 x 2 factorial design was used involving increments of .0690 DL-methionine from 0 to .3090, 0 or .20% choline chloride, and 0 or . 10% potassium sulfate. The variables were added to a 21 90 protein basal diet containing 61% ground yellow corn and 32% dehulled soybean meal to which no supplemental choline or sulfate was added. Each of the 24 diets was fed to two pens of medium-type turkeys of each sex (9 birds per pen) from four to eight-weeks of age in the first experiment, and to two pens of Large White turkeys of each sex (9 and 8 birds per pen for males and females, respectively) from three to seven-weeks of age in the second experiment. From the addition of .06% and .12% DL-methionine, body weight gains were increased 11.0 and 16.5% in the first experiment, and 8.9 and 13.690 in the second experiment, respectively. In addition, methionine increased feed consumption about 1/3 these amounts, and feed efficiencies 1/2 these amounts. The addition of choline or sulfate failed to significantly increase these parameters, even in the absence of methionine. No significant interactions were observed among the dietary variables in either experiment. / M. S.

Duodenal, jejunal and ileal sections from sheep were used to study <i>in vitro</i> absorption of valine, threonine and methionine. Everted sacs were incubated for 45 minutes at 39 C under an atmosphere of 95% O₂ - 5% CO₂ in Krebs-Ringer bicarbonate buffer (mucosal and serosal fluid) containing 5 μmoles per ml of the test amino acid. The hydrolyzed tissue and mucosal and serosal fluids were analyzed for amino acid content. The duodenum absorbed smaller quantities of amino acids from the mucosal fluid than the jejunum and significantly (P<.01) less than the ileum. The release of amino acids from duodenal tissue was small for threonine and methionine and negative for valine. Accumulation of amino acids by duodenal tissue was not significantly different from the ileum, but this represented a larger part of the amount absorbed from the mucosal fluid. The duodenum appears to be of limited importance as a site of amino acid absorption. The jejunum readily absorbed valine and methionine, but absorbed only a small amount of threonine from the mucosal fluid. The release of amino acids into the serosal fluid by the jejunum was very slight in comparison to the ileum (P<.01). Tissue accumulation of amino acids was significantly less (P<.05) than in either the duodenum or ileum. While the jejunum generally absorbed large amounts of amino acids from the mucosal fluid, it did not release or accumulate amino acids in large amounts. The ileum maximized movement of all amino acids. It absorbed significantly more (P<.01) amino acids from the mucosal fluid and released significantly more (P<.01) amino acids into the serosal fluid. The accumulation of valine and threonine by ileal tissue was significantly greater (P<.05) than the jejunum. The ileum, therefore, appears to be the most active and efficient site of valine, threonine and methionine absorption in the sheep. / Master of Science

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines. Most cells proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line MeWo. The MeWo-LC1 cell line has been shown to have a cellular phenotype similar to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin dependent enzymes methionine synthase and methylmalonyl-CoA mutase. Inability of cblC cells to complement the defect in cobalamin metabolism in MeWo-LC1 suggested that the defect was caused by decreased activity of the MMACHC gene product. However, no potentially disease causing mutations could be detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression could be detected in MeWo-LC1, and there was virtually complete methylation of a CpG island at the 5' end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation; the CpG island was partially methylated in MeWo and only lightly methylated in control fibroblasts. Transfection of MeWo-LC1 with wild type MMACHC with a constitutive promoter resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

No description available.

Except for tyrosine, the amino acid requirement of parenterally fed (PN) human neonates has not been derived. Methionine and cysteine are indispensable and dispensable sulphur amino acids respectively. Cysteine is synthesized from methionine. Cysteine is unstable in solution, and is left out or added in very small amounts to amino acid solutions. Methionine is added to compensate for the lack of cysteine, assuming that the neonate will convert methionine to cysteine to meet the body’s metabolic demand. Methionine is hepatotoxic and there is evidence that the neonate has limited ability for its conversion to cysteine. To determine the requirement of the neonate for methionine, PN-fed, stable, post-surgical neonates received graded intakes of methionine. The mean methionine requirement was estimated to be 49 mg.kg-1.day-1, which is 48 to 90% of the methionine content of current commercial amino acid solutions. Because cysteine is the rate limiting substrate for glutathione (GSH) synthesis and current methods of determining amino acid requirement measure requirement for protein synthesis, SAA requirements for maintenance of GSH status was deleniated in healthy adult males and in PN-fed human neonates. GSH kinetics was measured in healthy men receiving the mean methionine requirement and graded intakes of cysteine. GSH synthesis did not change with the addition of cysteine. Additionally, PN-fed post-surgical neonates recieved a methionine-adequate cysteine-free PN followed by cysteine supplemented PN for two 3-day periods and GSH kinetics measured on days 3 and 6. There was no change in GSH synthesis in response to cysteine supplementation. It is concluded that the PN-fed human neonate is capable of synthesizing enough cysteine from methionine not only for protein synthesis but for GSH synthesis. For both healthy men and stable post-surgical neonates, the requirement for GSH synthesis is met at the sulphur amino acid requirement derived using the indicator amino acid technique

Except for tyrosine, the amino acid requirement of parenterally fed (PN) human neonates has not been derived. Methionine and cysteine are indispensable and dispensable sulphur amino acids respectively. Cysteine is synthesized from methionine. Cysteine is unstable in solution, and is left out or added in very small amounts to amino acid solutions. Methionine is added to compensate for the lack of cysteine, assuming that the neonate will convert methionine to cysteine to meet the body’s metabolic demand. Methionine is hepatotoxic and there is evidence that the neonate has limited ability for its conversion to cysteine. To determine the requirement of the neonate for methionine, PN-fed, stable, post-surgical neonates received graded intakes of methionine. The mean methionine requirement was estimated to be 49 mg.kg-1.day-1, which is 48 to 90% of the methionine content of current commercial amino acid solutions. Because cysteine is the rate limiting substrate for glutathione (GSH) synthesis and current methods of determining amino acid requirement measure requirement for protein synthesis, SAA requirements for maintenance of GSH status was deleniated in healthy adult males and in PN-fed human neonates. GSH kinetics was measured in healthy men receiving the mean methionine requirement and graded intakes of cysteine. GSH synthesis did not change with the addition of cysteine. Additionally, PN-fed post-surgical neonates recieved a methionine-adequate cysteine-free PN followed by cysteine supplemented PN for two 3-day periods and GSH kinetics measured on days 3 and 6. There was no change in GSH synthesis in response to cysteine supplementation. It is concluded that the PN-fed human neonate is capable of synthesizing enough cysteine from methionine not only for protein synthesis but for GSH synthesis. For both healthy men and stable post-surgical neonates, the requirement for GSH synthesis is met at the sulphur amino acid requirement derived using the indicator amino acid technique