No description available.

A histochemical method for the localization of choline acetyltransferase, the synthesizing enzyme of acetylcholine has been recently developed. Conditions of the method were first investigated and optimized in the spinal cord of the rat where cholinergic neurones are known to be present. Following standardization of the method the localization of the enzyme was studied in the deep cerebellar nuclei. A series of transverse sections were stained to facilitate, allocation of cells demonstrating enzyme activity to their respective nuclei as described in the literature. The presence of choline acetyltransferase was used as a direct indication of a cholinergic neurone, and was found predominantly in the cells in the ventral portion of the lateral nucleus, on the border of the interpositus and lateral nuclei and in the caudal portion of the medial nucleus. The results obtained are consistent with other studies indicating that a portion of the cerebello-thalamic pathway is cholinergic. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

No description available.

No description available.

The overwhelming increase in antibiotic resistant bacterial strains poses a serious public health problem, with multiply-resistant strains becoming an important cause of mortality in hospitals. The predominant mechanism of resistance to aminoglycoside antibiotics involves enzymatic modification of the drug, rendering it ineffective. The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ii (AAC(6')-Ii) in complex with its cofactor, acetyl coenzyme A (AcCoA), was determined at 2.7 Å resolution by the multiwavelength anomalous diffraction technique. The resolution of this structure was subsequently extended to 2.15 Å by molecular replacement, with no significant changes in the topology of the complex. The enzyme was found to exhibit a novel CoA-binding fold, with the cofactor bound in a cleft between the N-and C-terminal arms of the protein molecule. Although the enzyme packs as a monomer in the I4₁32 crystal form, the most probable physiological dimer of the complex was determined through analysis of a number of symmetry-related molecules. The crystal structure of the AAC(6')-Ii•AcCoA complex was compared to the structures of three members of a large superfamily of GCN5-related 𝘕-acetyltransferases (GNATs), namely yeast histone acetyltransferase HAT1, 𝘕-myristoyltransferase, and aminoglycoside acetyltransferase(3)-Ia. Despite negligible sequence similarity between these GNAT superfamily members, a distinct folding pattern is conserved in all four structures. This establishes AAC(6')-Ii as a structural homolog of enzymes with protein acetylating activity, supporting the hypothesis that the enzyme may possess another physiological function in 𝘌𝘯𝘵𝘦𝘳𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘧𝘢𝘦𝘤𝘪𝘶𝘮. / Thesis / Master of Science (MS)

No description available.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: L-carnitine is a compound with a long history in biochemistry. It plays an important role in mammals, where many functions have been attributed to it. Those functions include the p-oxidation of long-chain fatty acids, the regulation of the free CoASH/ Acyl-CoA ratio and the translocation of acetyl units into mitochondria. Carnitine is also found in lower eukaryotic organisms. However, in contrast to the multiple roles it plays in mammalian cells, its action appears to be restricted to the transport of activated acyl residues across intracellular membranes in the lower eukaryotes. In the yeast Saccharomyces cere visiae , the role of carnitine consists mainly of the transfer of activated acetyl residues from the peroxisome and cytoplasm to the mitochondria. This process is referred to as the carnitine shuttle. This system involves the transfer of the acetyl moiety of acetyl-CoA, which cannot cross organellar membranes, to a molecule of carnitine. Subsequently, the acetylcarnitine is transported across membranes into the mitochondria, where the reverse transfer of the acetyl group to a molecule of free CoA occurs for further metabolism. Carnitine acetyl transferases (CATs) are the enzymes responsible for catalysing the transfer of the activated acetyl group of acetyl-CoA to carnitine as well as for the reverse reaction. In the yeast S. cerevisiae, three CAT enzymes, encoded by the genes CAT2, YAT1 and YAT2, have been identified. Genetic data suggest, that despite the high sequence similarity, each of the genes encodes for a highly specific activity that is part of the carnitine shuttle. So far, the specific function of any of the three CAT enzymes has been elucidated only partially. The literature review focuses mainly on the importance of the carnitine system in mammals. After discussing the discovery and biosyntheses of carnitine, the enzymatic background of and molecular studies on the carnitine acyltransferases are described. The experimental section focuses on elucidating the physiological roles and cellular localisation of the three carnitine acetyltransferase of S. cere visia e. We developed a novel enzymatic assay to study CAT activity in vivo. By C-terminal tagging with a green fluorescent protein, we localised the three CAT enzymes. However, all our genetic attempts to reveal specific roles for and functions of these enzymes were unsuccessful. The overexpression of any of the CAT genes could not cross-complement the growth defect of other CAT mutant strains. No phenotypical difference could be observed between strains carrying single, double and triple deletions of the CAT genes. Furthermore, the expression of the Schizosaccharomyces pombe dicarboxylic acid transporter can complement the deletion of the peroxisomal citrate synthase, but has no effect on the carnitine shuttle per se. Our data nevertheless suggest that Cat2p is the enzyme mainly responsible for the forward reaction, e.g. the formation of acetylcarnitine and free CoA-SH from acetyl-CoA and carnitine, whereas Yat1 pand Yat2p may be required mainly for the reverse reaction. / AFRIKAANSE OPSOMMING: L-karnitien is 'n verbinding met 'n lang geskiedenis in die biochemie-veld. Dit speel 'n belangrike rol in soogdiere, waar verskeie funksies daaraan toegeskryf word. Dié funksies sluit in die p-oksidasie van lang-ketting-vetsure, die regulering van die vrye KoA-SH-tot-asiel-KoA-verhouding en die oordrag van asetieleenhede na die mitochondria. Karnitien word ook in laer eukariotiese organismes gevind. In teenstelling met die verskeidenheid rolle wat dit in soogdierselle vervul, is die funksie in laer eukariote tot die transport van geaktiveerde asetielderivate oor intrasellulêre membrane beperk. In die gis Saccharomyces cerevisiae is die funksie van karnitien meestal beperk tot die vervoer van geaktiveerde asetielresidu's vanaf die sitoplasma en piroksisome na mitochondria, 'n proses wat as die "karnitiensiklus" bekend staan. Die proses behels die oordrag van die asetielgedeelte van asetiel-KoA, wat nie oor organelmembrane kan beweeg nie, na 'n molekuul van karnitien. Gevolglik word die asetielkarnitien oor die membraan na die mitochondria vervoer, waar - met die oog op verdere metabolisme - die omgekeerde oordrag van die asetielgroep na 'n vrye molekuul van KoA plaasvind. Karnitienasetiel-transferases (KAT's) is die ensieme wat verantwoordelik is vir die katalisering van die oordrag van die geaktiveerde asetielgroepe van asetiel-KoA na karnitien, sowel as vir die omgekeerde reaksie. In die gis S. cerevisiae is drie KAT-ensieme geïdentifiseer wat deur die gene CAT2, YAT1 en YAT2 gekodeer word. Genetiese data dui daarop dat, ten spyte van die hoë mate van homologie van die DNA-volgordes, elke geen vir 'n hoogs spesifieke aktiwiteit, wat deel van die karnitiensiklus is, kodeer. Tot dusver is die spesifieke funksie van die drie individuele KAT-ensieme net gedeeltelik ontrafel. Die literatuurstudie fokus hoofsaaklik op die belangrikheid van karnitiensisteme in soogdiere. Na 'n bespreking van die ontdekking en biosintese van karnitien, word die ensimatiese agtergrond en molekulêre studies van KAT's beskryf. Die eksperimentele deel konsentreer op die ontrafelling van die fisiologiese rol en intrasellulêre lokalisering van die drie KAT-ensieme van S. cerevisiae. Eerstens is 'n nuwe ensimatiese toets ontwikkel om KAT-aktiwiteit in vivo te bestudeer. Deur C-terminale aanhegting van 'n groen fluoreserende proteïen kon die drie KATensieme gelokaliseer word. Daar kon egter nie met behulp van genetiese studies verder lig gewerp word op die spesifieke rolle en funksies van hierdie KAT-ensieme nie. Die ooruitdrukking van enige van die KAT-gene kon nie die groeidefek van ander KAT-mutantrasse kruiskomplementeer nie. Geen fenotipiese verskil tussen rasse wat 'n enkel, dubbel of trippel delesie van die KAT-gene bevat, kon waargeneem word nie. Verder kon die uitdrukking van Schizosaccharomyces pombe se dikarboksielsuurtransporter die delesie van die peroksisomale sitraatsintetase komplementeer, maar het dit as sulks geen effek op die karnitiensiklus gehad nie. Die data wat deur hierdie studie verkry is, dui nogtans daarop dat Cat2p die ensiem is wat hoofsaaklik verantwoordelik is vir die voorwaartse reaksie, met ander woorde die vorming van asetielkarnitien en vrye KoH-SH van asetiel-KoA en karnitien, terwyl Yat1 p en Yat2p hoofsaaklik vir die omgekeerde reaksie benodig word.

No description available.

This thesis contains the synthesis of a series of novelligands that include the enantiomerically pure 2-oxazoline moiety. The thesis also considers the application of new nucleophiles for the palladium catalysed allylic substitution reaction. The synthesis of enantiomerically enriched analogues of y-amino butyric acid (GABA) is presented. The first series of ligands are designed as analogues of acetyl acetonate (acac). The ligands include the enantiomerically pure 2-oxazoline ring and a carbonyl moiety. The ligands are available in good yield in two steps. The second series of ligands include a ligating sulfur atom. The synthesis of novel oxazoline-sulfide ligands is detailed. The diastereoselective oxidation of these ligands is considered. Diastereomerically pure oxazoline-sulfoxide ligands are prepared in good yield. New nucleophiles are applied to the palladium catalysed allylic substitution reaction. The substitution products are available in good yield and with excellent stereoselectivity. The synthesis of analogues of GABA is considered. The preparation of enantiomerically enriched a-substituted-y-amino butyric acids is presented. The stereocentre is introduced in the first step of the synthesis. The analogues are subsequently isolated in good yield after six steps.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Using vegetable oils directly as an alternative biofuel presents several problems as such oils typically possess poor fuel qualities including high viscosity, low volatility, and poor cold temperature properties. The ornamental shrub Euonymus alatus produces unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) that have an acetyl group in the sn-3 position instead of a long chain fatty acid. The presence of this sn-3 acetyl-group give acetyl-TAGs properties desirable for biofuels, such as reduced viscosity, comparted to the normal long chain triacyglycerols found in most vegetable oils. Acetyl-TAGs are synthesized by the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) and Euonymus fortunei diacylglycerol acetyltransferase (EfDAcT) enzymes. Both enzymes catalyze the transfer of an acetyl group from acetyl-CoA to diaclglycerol (DAG) to produce acetyl-TAGs. Previous work demonstrated that expression of EaDAcT combined with the suppression of a diacylglycerol aceyltransferase (DGAT1) in Camelina sativa led to seeds with 85 mol % acetyl-TAGs. Increasing acetyl-TAG levels further was explored using two strategies. Over expression of citrate lyase to increase the pool of acetyl-CoA to be used as a substrate for the acetyltransferase enzymes failed to increased levels of acetyl-TAGs. A second approach involved expressing EfDAcT in Camelina sativa. EfDAcT has demonstrated higher activity in vitro and in vivo and its expression in yeast leads to approximately 50 % higher levels of acetyl-TAGs compared to EaDAcT. The expression of EfDAcT coupled with the suppression of DGAT1 in Camelina sativa resulted in 90 mol % acetyl-TAGs in the transgenic seeds. Levels of EfDAcT protein analyzed in developing transgenic Camelina sativa seeds across a 40 day time period were highest at 15 and 20 days after flowering. Following these time points acetyl-TAG accumulation increased rapidly, coinciding with the higher enzyme expression levels. The optimization of additional promoters to ensure expression of EfDAcT in the last half of seed development could represent another way to further increase acetyl-TAGs in the future.