京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18866号 / 医博第3977号 / 新制||医||1008(附属図書館) / 31817 / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸井 雅和, 教授 平岡 眞寛, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Contrary to a previous report, S-adenosyl-L-methionine (SAM) affords stable solutions in D2O and the 1H NMR spectrum can be determined. Comparison with the spectra of the model compounds adenosine, L-methionine and L-methionine-S-methyl sulfonium iodide allows complete assignment of the proton resonances. Coupling constants were determined by homonuclear decoupling and graphical analysis and were confined by computer simulation. Details of the molecular conformation were determined by application of the Karplus equation and calculation of relative rotational isomer populations. Evidence indicates that the ribose ring is puckered preferentially in the C3'-exo conformation and that the C4'-C5' bond is constrained to a rotamer in which the sulfonium center is gauche to H4'. No conformational constraints were detected for the Cα-Cβ and Cβ-Cα bonds of the methionine side chain. The purine ring was shown to be oriented preferentially anti by intermolecular association studies with adenosine 5'-phosphate in the presence of MN(II). Spectra of samples of (-)S-adenosyl-L-methionine of biological origin, differing in activity, counter ion and commercial source, have consistently revealed the presence of a small amount of the (+) sulfonium diastereomer. Arguments are presented to explain the failure of previous workers to detect (+)S-adenosyl-L-methionine in biological preparations.

Rumen protected methionine has been used in an effort to improve the amino acid composition of metabolisable protein since the early 1960’s. The positive response in dairy cows in terms of milk protein composition and milk production, especially during early lactation has been well documented. Rumen protected methionine supplementation contributes to improving the protein efficiency of the dairy cow which improves the overall productivity of the dairy enterprise. Recently a locally developed liquid rumen protected methionine prototype became available. In our study this product was evaluated through a series of experiments in conjunction with two standard, well known methionnine sources, Smartamine ™ M and unprotected DL-methionine that provided a reference to the relative bioavailability of the liquid rumen protected methionine. In the first of the two studies the effect of methionine supplementation on milk yield, milk composition as well as milk protein composition was evaluated through the milk composition technique. The ability of the liquid rumen protected methionine to elevate blood plasma methionine levels was also evaluated through the blood plasma technique after oral dosing and post ruminal infusion of methionine. The liquid rumen protected methionine prototype induced no response in either milk yield or milk composition. Results suggested that the prototype is either not adequately protected against rumen degradation or it is not available for absorption in the small intestine. The inability of the liquid rumen protected methionine prototype to elevate blood plasma methionine after post ruminal infusion further proved that the product is not available for absorption at this site either. In the event that the product’s mode of action or method of protection caused it not to be detected as pure methionine in the blood, an effect on milk yield would have been expected which was not the case. This product proved to have a very low or no bioavailability in comparison to the well researched and proven Smartamine ™ M. / Dissertation (MSc(Agric))--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted

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

We investigated the transfer of three antioxidants - melatonin, S-adenosyl methionine (SAM) and various forms of vitamin E - across the term, normal human placenta. The transport technique involved the single, isolated placental cotyledon system in vitro. Melatonin crossed the placenta rapidly, equally to the freely diffusible marker, antipyrine. There was no biotransformation of the agent. SAM was transferred slowly, similarly to passively transported L-glucose as a marker. There was a breakdown of SAM to at least one other derivative; the process appeared to be nonenzymatic. Vitamin E was transferred slowly, at a rate only 10% of L-glucose. The natural RRR (nonracemic) form of vitamin E was transported best. Free vitamin, rather than the acetate seems to be transferred best, a finding that will require further study.

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