Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

Chemical, physical and sensory properties of omega-3 fatty acid enriched chicken frankfurters developed with flax oil and microencapsulated fish oil at 1.2%, 2.4% and 3.6% were evaluated. Four replicate batches of frankfurters were produced for texture profile analysis and TBARS for assessment of lipid oxidation over four weeks of refrigerated storage. Gas chromatograph analysis indicated that omega-3 fatty acid levels increased (p<0.05) with flax and fish oils treatments resulting in a shift in omega-6/omega-3 with no increase in lipid oxidation over the storage period. The two highest levels of fish oil resulted in increased redness, hardness, gumminess and chewiness (p<0.05) with the highest fish oil having the lowest rating for acceptability. 1.2 and 2.4% flax oil and 1.2% fish oil samples were softer and juicier than commercial frankfurters. Addition of oils high in omega-3 fatty acids to chicken-based frankfurters can result in product resistant to oxidation and acceptable to consumers.

The aim of this research was to isolate and characterize novel strains of marine protists with potential to commercially produce PUFAs. Twelve trips were made visiting 10 different locations in the Canadian Maritime Provinces. Sixty-nine strains were isolated and screened for biomass and fatty acid production. Those meeting specific criteria were selected for further investigation including characterization by 18S rDNA sequencing. Isolate ONC-KTB-56 produced the greatest amount of biomass (1 807 mg L-1) and fatty acids (24.6% dry weight). Of the total fatty acids, ARA, EPA and DHA comprised 0.89, 1.22 and 4.7 percent, respectively. Isolate ONC-KTB-14 produced 1 704 mg L-1 dry biomass with 5.4 percent fatty acids including 1.44, 1.35 and 37.5 percent, ARA, EPA and DHA, respectively. Through optimization of culture conditions biomass, fatty acid content and the proportions of specific fatty acids can be increased. With such optimization, there is potential for isolates ONC-KTB-14 and ONC-KTB-56 to be grown at a commercial scale for production of PUFAs.

The clearance of circulating glucose and long-chain fatty acids (FA) into skeletal muscle involves the translocation of glucose transporter GLUT4, fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm) and fatty acid transport protein (FATP) 1 and 4 to the plasma membrane (PM). FAT/CD36 also appears to participate in the regulation of mitochondrial FA oxidation. Metabolic challenges are known to increase FA transport and/or oxidation, but whether this is solely attributable to the translocation of FAT/CD36 to the sarcolemma and/or mitochondria is unknown. Moreover, the signaling and trafficking pathways involved in the translocation of FA transporters are largely unexplored. In this thesis it was found that FA transport was markedly increased following insulin (+2.9-fold) or contraction (+1.7-fold) stimulation of skeletal muscle, along with the PM contents of FAT/CD36 (+78%, +55%,), FABPpm (+61%, +62%), FATP1 (+84%, +61%) and FATP4 (+60%, +66%) (p<0.05). Upon combining the two stimuli, only the translocation of FAT/CD36 (+179%) and FATP1 (+125%) to the PM was additive, suggesting that these transporters may reside in distinct insulin-sensitive and contraction-sensitive intracellular compartments. The translocation of FA transporters may involve the insulin-signaling protein Akt2. It was found that insulin-stimulated FA transport and PM translocation of FA transporters was essentially prevented in Akt2 knockout mice. Following contraction, FA transport was also markedly blunted, along with an impaired translocation of both FAT/CD36 and FATP1, but not FABPpm or FATP4. FA oxidation and mitochondrial FAT/CD36 appearance were also inhibited following muscle contraction in knockout mice (p<0.05). Whether the GLUT4 trafficking protein Munc18c is important for the vesicular re-distribution of FA transporters to the PM or mitochondria was also investigated. FA uptake was comparably increased 1.4 fold with insulin and contraction in both wildtype and heterozygous Munc18c-/+ mice, as were PM FA transporters FAT/CD36 (+82%, +84%), FABPpm (+39%, +43%), FATP1 (+40%, +38%) and FATP4 (+33%, +32%) (p<0.05). Contraction-stimulated mitochondrial FA oxidation was also increased similarly in wildtype (+39%) and Munc18c-/+ mice (+33%). These studies demonstrate that a number of FA transporters are involved in upregulating skeletal muscle FA transport, although their signaling and trafficking pathways may differ from that of GLUT4.

Peanut (Arachis hypogaea L.) has the potential to become a major source of biodiesel but for market viability, peanut oil yields must increase and specific quality requirements must be met. Oil yield in peanut is influenced by many components, including oil concentration, seed mass, and mean oil produced per seed. All of these traits can be improved through selection as long as there is sufficient genetic variation. Thus, elucidating the genetics of oil concentration, seed mass, and mean oil produced per seed in peanut is essential to advancing the development of genotypes with high oil yields. Additive genetic effects were predominant for oil concentration in two generation means analyses involving a proprietary high oil breeding line and additive genetic variance was highly significant in a complete four-parent diallel analysis. Genetic variance for weight of 50 sound mature kernels (50 SMK) and mean oil produced per SMK (OPS) was additive the diallel analysis. Narrow-sense heritability estimates were high for oil concentration in both the diallel and generation means analyses. Narrow-sense heritability was also high for 50 SMK, but was low for OPS. The low OPS heritability estimate was caused by the negative correlation between oil concentration and seed mass. Consequently, oil concentration and seed mass can be improved through early-generation selection, but large segregating populations from high oil crosses will be needed to identify progeny with elevated oil concentrations that maintain acceptable seed sizes. Increasing the ratio of oleic to linoleic acid (O/L) in peanut oil and reducing the long chain saturated fatty acid concentration (which includes arachidic, behenic, and lignoceric acids) produces high quality, stable methyl esters for biodiesel. Therefore, elucidating the inheritance of these factors and their relationships in peanut populations segregating for high oil is critical. The results from generation means analysis confirm that the high-oleic trait is under simple genetic control and can be manipulated through selection. Oil concentration was negatively correlated with oleic acid concentration in the F2 generations of both crosses and positively correlated with arachidic acid in most of the segregating generations that were evaluated. Therefore, developing a peanut genotype high in oil and oleic acid concentration that has reduced long chain saturates will require the evaluation of large numbers of segregating progeny.

No description available.

An adequate ingestion of essential fatty acids is required for optimal development of the central nervous system and visual acuity in infants. For breast feeding mothers, it is important that a diet containing an adequate balance of essential fatty acids of the n-6 and n-3 series be consumed as this is reflected in breast milk. The objective of this investigation was to determine the essential fatty acid status of breast feeding women and their infants in Havana, Cuba, with particular focus on the n-3 series. The group of 56 Cuban mothers and infants under investigation did not show biochemical or functional signs of poor essential fatty acid status. Based on the biochemical and functional data collected, it is conceivable to conclude that n-3 fatty acid deficiencies must be exceedingly rare, if they exist at all, in the population of breast feeding women and their infants residing in Havana, Cuba. (Abstract shortened by UMI.)

Narwhals (Monodon monoceros) are sentinel species in the Arctic environment and are a vital component for Inuit culture and subsistence. The Arctic is undergoing rapid changes in temperature and sea ice cover and relatively little is known about how this has and will change narwhal foraging behaviour. There are three narwhal populations in the world, the Baffin Bay (BB), Northern Hudson Bay (NHB), and East Greenland (EG) populations; however, foraging behaviour, in terms of dive behaviour and primary dietary components, has really only been investigated in the BB population. Using a combination of stable isotopes, fatty acids, genetic techniques, and satellite tracking technologies I evaluated foraging behaviour in all three of the world’s narwhal populations. I also investigated social structure in the BB population to determine how adaptable narwhals are to a changing and dynamic Arctic environment. Stable isotopes (δ13C and δ15N) and fatty acids are chemical signatures in the tissues of an organism that can provide long-term information on their diet over varying temporal scales depending upon the tissue. Stable isotope analysis in the three narwhal populations found they forage on different primary prey, suggesting narwhal are adaptable in their preferred prey and that there is potential for them to adjust foraging behavior in the face of changing climate. Dietary changes were also assessed over three decades to determine how sea ice changes have affected narwhal foraging for the NHB and BB populations. Dietary changes were evident and can be attributed to changes in sea ice patterns and an altered migratory pathway for narwhals. An understanding of narwhal social structure is also needed to determine how behaviourally flexible narwhal are in diet and site fidelity. Genetic relatedness and dietary signatures from fatty acids were assessed for an entrapped group to determine if individuals that are closely related forage together, which would support a matrilineally driven social structure where females teach their young foraging strategies, and/or travel and forage together. I found no evidence that narwhals form a matrilineal social group, but they may display a fission-fusion structure, which may be an adaptation to patchy prey distribution in the Arctic. Finally, narwhal dive behaviour in all three populations was investigated to determine if dive behaviour could be used to predict diet. Dive differences among populations did correspond with differences in diet, suggesting that narwhals employ specialized foraging strategies. This has repercussions for their ability to adapt to ecosystem changes. Overall, narwhals may be more flexible in terms of their foraging behaviour than previously believed. However, an increased resilience to changing food webs will not be the only predictor of how narwhals will fare in the face of a changing climate; how they respond to increased industrial activities in their preferred habitats, increased predation from southern predators, and increased competition from southern cetaceans and humans alike, will play an equally large role in how they cope with the future.

Strawberry (Fragaria ananassa) is an economically important soft fruit that is highly valued as a fresh product and flavouring. During ripening, strawberry fruits undergo a number of physiological changes affecting colour, texture and flavour. An understanding of these changes at the biochemical and molecular level will be important in developing strategies for enhancing the quality attributes of this fruit. A cDNA encoding a ripening- enhanced acyl carrier protein (RE-ACP) was previously isolated from strawberry fruit. AC? is an essential component of fatty acid synthesis in both plants and animals. The aims of this thesis were to isolate and characterise this and other members of the ACP multigene family expressed in strawberry fruit. Six closely related putative AC? cDNA isoforms were identified from strawberry. Two of these were obtained by screening a cDNA library from ripe fruit and three were obtained by a technique known as candidate fragment length polymorphism (CFLP) that utilised ACP gene-specific primers for AFLP-cDNA display. Northern analysis was not able to differentiate their expression but ACP was highly up-regulated in ripening fruit whereas low levels of expression were detected in other strawberry tissues, including achenes (seeds), expanding leaves and flowers. The RE-ACP was over-expressed in E. coli and the recombinant protein partially purified. The over-expressed protein had a M(_r) of 20kDa on SDS-PAGE and appeared to form a dimer. A genomic library was constructed from F. ananassa from which two different genomic clones closely related to RE-ACP were obtained. Promoter analysis indicated the presence of regulatory elements. The characterization of putative ACP cDNA and genomic clones, including the 5' upstream regions, is described and their possible role in strawberry fruit is discussed. Key words: Strawberry, fruit, ripening, gene expression, genomic, cDNA, fatty acid, acyl carrier protein, aroma, promoter.

Thesis (M.Sc. (Nutrition))--Mahidol University, 1982.