Fatty acid biosynthesis from (1-$ sp{14}$C) acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at 82.3 nmol/hr/mg protein in the presence of 200$ mu$M acetate, 0.5mM each of NADH, NADPH and CoA, 6mM each of ATP and MgCl$ sb2$, 1mM each of MnCl$ sb2$ and glycerol-3-phosphate (G3P), 15mM KHCO$ sb3$, and 0.1M Bis tris propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear for up to 6 hours with 80 to 120 $ mu$g/ml plastid protein. ATP and CoA were absolute requirements, whereas divalent cations, potassium bicarbonate and reduced nucelotides all improved activity by 2 to 10 fold. Mg$ sp{2+}$ and NADH were the preferred cation and nucleotide, respectively. G3P and dihydroxyacetone phosphate had little effect, and dithiothreitol and detergents generally inhibited incorporation of $ sp{14}$C-acetate into fatty acid. / Glycerolipid synthesis was obtained from $ sp{14}$C-acetate, (U-$ sp{14}$C) G3P and (U-$ sp{14}$C) glycerol at relative rates of 3.7:1.0:0.1, respectively. (Abstract shortened by UMI.)

Differences in fatty acid distributions in adipose tissue and fatty acid synthetase levels in the liver were determined in Strain A/ST mice of different ages and diets. Since fatty acids have been found to be influential in many disease processes such as heart disease and cancer, which become more prevalent with increasing age, it is important to understand the processes of fat metabolism and changes that occur during the life-stage of senescence. Fatty acid distributions were determined by gas liquid chromatography and fatty acid synthetase (FAS) activities by spectrophotometry.The data from FAS analyses indicated that the mice fed the highfat palmitic acid and low-fat corn oil diets were similar to previous research. The mice fed the stearic acid diets had FAS activity that was affected in a very different manner than other high-fat diets.The results of this study also indicated that aging does not significantly effect the distribution of fatty acids in the adipose tissue of experimental mice. Weight gain in the middle age mice appears to be the result of an increase in all types of fatty acids and not just increased storage of one or a few types. / Department of Biology

Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

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by Kwan Kwok Yiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 145-155). / Acknowledgment --- p.i / Abstract --- p.ii / List of Tables --- p.vii / List of Figures --- p.x / List of Abbreviations --- p.xii / Chapter Chapter 1 --- Literature review / Chapter 1.1 --- Historical background --- p.1 / Chapter 1.2 --- Chemistry of trans and cis fatty acids --- p.3 / Chapter 1.3 --- Dietary source of trans fatty acids --- p.6 / Chapter 1.4 --- Consumption of trans fatty acids among Western countries --- p.9 / Chapter 1.5 --- Current health concern for excessive intake of trans fatty acids --- p.10 / Chapter 1.6 --- Metabolism of trans fatty acids --- p.13 / Chapter 1.6.1 --- Absorption --- p.15 / Chapter 1.6.2 --- Oxidation --- p.15 / Chapter 1.6.3 --- Incorporation --- p.16 / Chapter 1.6.4 --- Selectivity --- p.17 / Chapter 1.7 --- Impact of trans fatty acids on essential fatty acid metabolism --- p.19 / Chapter 1.8 --- Desaturation and elongation of trans fatty acids --- p.21 / Chapter 1.9 --- Trans fatty acids and neonatal growth --- p.23 / Chapter Chapter 2 --- Amount of trans fatty acids in Hong Kong fast foods / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Objective --- p.25 / Chapter 2.3 --- Materials and methods --- p.26 / Chapter 2.4 --- Results --- p.27 / Chapter 2.5 --- Discussion --- p.31 / Chapter Chapter 3 --- Cross-cultural study of trans fatty acids in human milk / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Objective --- p.35 / Chapter 3.3 --- Materials and methods --- p.36 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Dietary information --- p.38 / Chapter 3.4.2 --- Fatty acid composition of Chinese and Canadian human milk --- p.40 / Chapter 3.4.3 --- Difference between Chinese and Canadian human milk --- p.40 / Chapter 3.4.4 --- Difference between Hong Kong and Chongqing Chinese human milk --- p.43 / Chapter 3.4.5 --- The change in milk fat and LCPUFA as lactation progresses --- p.43 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Trans fatty acids in human milk --- p.46 / Chapter 3.5.2 --- Content of LCPUFA in human milk --- p.47 / Chapter 3.5.3 --- Content of 18:2n-6 in human milk --- p.48 / Chapter 3.5.4 --- Fat content in Hong Kong and Chongqing Chinese human milk --- p.49 / Chapter 3.6 --- Conclusion --- p.50 / Chapter Chapter 4 --- Trans fatty acids and maternal and neonatal essential fatty acid metabolism / Chapter 4.1 --- Introduction --- p.51 / Chapter 4.2 --- Objectives --- p.53 / Chapter 4.3 --- Materials and methods --- p.53 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Experiment1 / Chapter 4.4.1.1 --- Relationship between the trans fatty acids in maternal diet and those in milk --- p.64 / Chapter 4.4.1.2 --- Relationship between the trans fatty acids in maternal diet and those in neonatal liver --- p.64 / Chapter 4.4.1.3 --- Content of 20:4n-6 in milk and in neonatal liver relative to that in maternal diet --- p.72 / Chapter 4.4.2 --- Experiment2 / Chapter 4.4.2.1 --- Amount of trans fatty acids in rat milk --- p.75 / Chapter 4.4.2.2 --- Trans fatty acids in rat liver phospholipids --- p.75 / Chapter 4.4.2.3 --- Linoleic acid (18:2n-6) content in rat and its relation to maternal diets --- p.86 / Chapter 4.4.2.4 --- Content of 20:4n-6 in rat milk --- p.86 / Chapter 4.4.2.5 --- Content of20:4n-6 in rat liver --- p.89 / Chapter 4.4.2.6 --- Suppression of the synthesis of 20:4t isomers in maternal and neonatal liver --- p.89 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Relationship between fatty acid composition of diet and that of milk --- p.93 / Chapter 4.5.2 --- 20:4n-6 in rat milk --- p.95 / Chapter 4.5.3 --- Transfer of trans fatty acids from maternal diet to neonatal liver phospholipids --- p.98 / Chapter 4.5.4 --- The inhibitory effect of trans fatty acids on synthesis of 20:4n-6 in neonatal liver --- p.99 / Chapter 4.5.5 --- Effect of 18:2n-6 supplement on 20:4n-6 level of neonatal liver --- p.101 / Chapter 4.5.6 --- Suppression of 18:2n-6 supplement on synthesis of 20:4t isomers --- p.101 / Chapter 4.6 --- Conclusion --- p.104 / Chapter Chapter 5 --- Accumulation and turnover of trans fatty acids / Chapter 5.1 --- Introduction --- p.105 / Chapter 5.2 --- Objective --- p.105 / Chapter 5.3 --- Materials and methods --- p.106 / Chapter 5.4 --- Results / Chapter 5.4.1 --- Accumulation of trans fatty acids in liver and adipose tissue --- p.108 / Chapter 5.4.2 --- Selectivity of individual 18:2 trans isomersin liver and adipose tissue --- p.112 / Chapter 5.4.3 --- Turnover of trans fatty acids --- p.112 / Chapter 5.4.4 --- Accumulation and turnover of 18:lt in brain --- p.115 / Chapter 5.5 --- Discussion / Chapter 5.5.1 --- Accumulation of trans fatty acids in liver and adipose tissue --- p.120 / Chapter 5.5.2 --- Turnover of trans fatty acids --- p.122 / Chapter 5.5.3 --- Accumulation and turnover of trans fatty acidsin brain --- p.124 / Chapter 5.6 --- Conclusion --- p.125 / Chapter Chapter 6 --- In vivo Oxidation of trans fatty acids in rat / Chapter 6.1 --- Introduction --- p.126 / Chapter 6.2 --- Objective --- p.127 / Chapter 6.3 --- Materials and methods --- p.127 / Chapter 6.4 --- Results --- p.129 / Chapter 6.4.1 --- Apparent oxidation of saturated fatty acids --- p.136 / Chapter 6.4.2 --- Apparent oxidation of 18:lt relative to 18:ln-9 --- p.136 / Chapter 6.4.3 --- Oxidation of 18:2t isomers relative to 18:2n-6 --- p.137 / Chapter 6.4.4 --- Effect of 18:2n-6 supplement in PHCO diet on oxidation per se --- p.137 / Chapter 6.5 --- Discussion --- p.138 / Chapter 6.5.1 --- Oxidation of 18:lt and 18:2t isomers --- p.139 / Chapter 6.5.2 --- Effect of 18:2n-6 supplement on oxidation per se --- p.140 / Chapter 6.6 --- Conclusion --- p.141 / General conclusion --- p.142 / References --- p.145

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The cardiac isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid (FA) uptake; thus increased ACCβ activity decreases fatty acid utilization thereby potentially leading to intracellular myocardial lipid accumulation and insulin resistance (IR). Previous studies show that greater flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of IR. In light of this, we hypothesize that increased HBP flux induces ACCβ gene expression thereby contributing to the onset of IR. Our initial work focused on ACCβ gene promoter regulation and suggest that the HBP modulates upstream stimulatory factor 2 (USF2) thereby inducing ACCβ gene expression. Here, we further investigated HBP-mediated regulation of ACCβ gene expression by transiently transfecting cardiac-derived H9c2 cells with an expression vector encoding the rate-limiting HBP enzyme (GFAT) ± the full length ACCβ and 4 truncated promoter-luciferase constructs, respectively. GFAT overexpression increased ACCβ gene promoter activity for the full length and 3 larger deletion constructs (p<0.001 vs. controls). However, GFAT-mediated and USF2-mediated ACCβ promoter induction was blunted when co-transfected with the -38/+65 deletion construct suggesting that USF2 binds to the proximal promoter region (near start codon). Further investigation proves that USF2 binds to ACCβ promoter and activates it, but that USF2 is not O-GlcNAc modified even though there is a strong correlation between increased O-GlcNac levels and USF2 activation of ACCβ. This would suggest that there is another O-GlcNac modified factor involved in this regulatory pathway. Our study demonstrates that increased HBP flux induces ACCβ gene promoter activity via HBP modulation of USF2. We propose that ACCβ induction reduces fatty acid oxidation, thereby leading to intracellular lipid accumulation (FA uptake>>FA oxidation) and the onset of cardiac IR. / AFRIKAANSE OPSOMMING: Die kardiale isoform van asetiel-CoA karboksilase (ACCβ) produseer maloniel-CoA, ‘n kragtige inhibeerder van mitochondriale vetsuur (VS) opname, en om hierdie rede sal verhoogde ACCβ aktiwiteit, vetsuur gebruik verlaag en potensieël aanleiding gee tot intrasellulêre miokardiale lipiedophoping en insulienweerstand (IW). Vorige studies toon dat groter fluks deur die heksosamienbiosintetiese weg (HBW) bydra tot die ontwikkeling van IW. In die lig hiervan hipotetiseer ons dat verhoogde HBW fluks, ACCβ geenuitdrukking induseer, en sodoende tot die onstaan van IW bydra. Ons aanvanglike werk het op ACCβ geenpromotorregulering gefokus, en voorgestel dat die HBW die opstroom stimuleringsfaktor 2 (USF2) moduleer en dus ACCβ geen uitdrukking induseer. Hier het ons verder die HBW-gemedieërde regulering van ACCβ-geenuitdrukking deur kortstondige tranfeksie van kardiaalverkrygde H9c2 selle met ‘n uitdrukkingsvektor wat kodeer vir die tempo-bepalende HBW ensiem (GFAT) ± die volle lengte ACCβ, en vier afgestompte promotor-lusiferase konstrukte onderskeidelik, te ondersoek. GFAT ooruidrukking het ACCβ geenpromotor aktiwiteit vir die volle lengte, en drie groter uitwissingskonstrukte verhoog (p<0.001 vs. kontrole). Hoewel GFAT- en USF2-gemedieërde ACCβ promotorinduksie tydens ko-transfeksie van die -38/+65 uitwissingskonstruk versag was, is dit voorgestel dat USF2 aan die proksimale promotor area (naby die beginkodon) bind. Verdere ondersoek bewys ook dat USF2 aan die ACCβ promotor bind en dit aktiveer, maar dat USF2 nie O-GlcNAc gemodifiseer word nie ten spyte van ‘n sterk korrelasie tussen verhoogde O-GlcNac vlakke en USF2 aktivering van ACCβ. Dit kan dus voogestel word dat daar ‘n alternatiewe O-GlcNac gemodifiseerde faktor betrokke is in hierdie reguleringsweg. Ons studie demonstreer dat verhoogde HBW fluks ACCβ geenpromotor aktiwiteit via HBW modulering van USF2 veroorsaak. Ons stel voor dat ACCβ induksie vetsuuroksidasie verlaag en so tot intrasellulêre lipiedophoping (VS opname >> VS oksidasie) en die onstaan van kardiale IW lei.