Oat is known to synthesize several epoxy fatty acids in seeds using peroxygenase (PXG), a type of hydroperoxide-dependent epoxygenase. This thesis aims to molecularly clone and functionally characterize the PXG genes from oat developing seeds. The research started with identifying additional PXG genes from oat expressed sequence tag (EST) databases using a previously identified oat peroxygenase AsPXG1 as a query sequence. This resulted in the identification of six homologous contig sequences from the EST data bases. Of them, two contigs with high sequence similarity and alignment with plant PXG/caleosin proteins were selected for cloning and functional analysis. Rapid amplification of cDNA ends (RACE) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to retrieve the full length cDNAs of the contigs, which resulted in identification of three putative PXG genes, AsPXG2, AsPXG3 and AsPXG4. Open reading frame (ORF) of AsPXG2 is 702 bp long encoding a polypeptide of 233 amino acids, while ORFs of both AsPXG3 and AsPXG4 are 627 bp in length coding for 208 amino acids. All these putative peroxygenases comprise a single transmembrane domain, presumably for lipid droplet anchoring, conserved hisditine residues for heme-binding and a conserved EF-hand motif for calcium-binding. To functionally characterize the three genes, their ORFs were individually expressed in Escherichia coli/Pichia pastoris. The enzymatic assays showed that all transformants produced 9,10-epoxystearic acid methyl ester in the presence of oleic acid methyl ester and cumene hydroperoxide, indicating all three genes encode functional peroxygenase. AsPXG3 has the highest specific activity at 42 mol/mg/min with about 25% substrate conversion efficiency. Substrate specificity assays on free fatty acids showed that AsPXG3 could epoxidize all mono- and poly-unsaturated fatty acids tested, with linolenic acid (C18:3-9c,12c,15c) being the most preferred substrate. Site-directed mutagenesis of three conserved histidines and nine conserved residues surrounding the histidines in AsPXG3 showed that substitution of the first conserved histidine at position 32 (H1) and the third conserved histidine at position 102 (H3) with alanine respectively resulted in complete loss of the enzymatic activity, while substitution of the second conserved histidine at position 98 (H2) resulted in only slight reduction of the activity, indicating that only H1 and H3 are absolutely essential and probably involved in heme-binding for the peroxygenase. Substitution of leucine at position 29 (M1), isoleucine at position 97 (M5), and lysine at position 101 (M8) with alanine reduced the enzymatic activity on oleic acid methyl ester by more than 80% relative to the wild type enzyme, indicating these three residues are also very important for catalytic activity. The activity of M1, M5 and M8 mutants was also drastically reduced on all other free mono-unsaturated fatty acids tested (>60%). However, to linolenic acid, M5 showed only slight reduction of the activity (~15%) and M8 even increased the activity by 12% relative to the wild type enzyme. These results suggest that these conserved residues might play roles in defining the shape and size of the catalytic site for interaction of the heme with fatty acid substrates.

No description available.

No description available.

No description available.

No description available.

No description available.

This Ph.D. dissertation presents results that shed light on whether there is an association between dietary intake and depressive disorder, feelings of sadness, or thoughts of death. This question is investigated in the context of a secondary data analysis using cross-sectional data from the National Health and Nutrition Examination Survey (NHANES) 1999-2002. Subjects in this study were identified as a subpopulation of the NHANES survey that completed the Composite International Diagnostic Interview questionnaire for major depression. The presence of depression may lead to a decline in dietary intake and nutritional status. Conversely, poor dietary intake may have a role in the etiology of depression. Specifically, depression is associated with lower plasma, erythrocyte, phospholipid, and adipose tissue omega-3 fatty acids. Little is known about the comprehensive dietary intake of those with depression or depressive symptoms. Initially, comparisons of the dietary profile between those with and without depression or depressive symptoms were investigated. Secondly, analysis was done to determine the relationship between dietary omega-3 fatty acids and depression or depressive symptoms. Lastly, to clarify the relationship between diet and depression, misreporting of dietary energy intake was evaluated.No significant differences in the dietary profile or nutritional biochemistries were found between the altered mood and normal mood groups. No significant differences were found in dietary omega-3 fatty acids between the depressed and non-depressed groups, even after adjusting for confounding variables. Misreporting of energy intake was not significantly associated with altered mood status and remained non-significant after adjusting for confounding variables. In both groups, a significant decreasing trend in the ratio of energy intake to estimated basal metabolic rate (EI:BMRest) was found as body mass index increased.Based on dietary intake alone, the relationship between depression and specific nutrients may not be apparent. Dietary intake among those with depression and depressive symptoms appears to be adequate, but adequacy may not be sufficient as a route for prevention or management of depression. Further investigation of diet and depression ought to measure diet, including nutrition supplements, and biochemical levels of nutrients from a broader age range of adults and children and those with comorbid illnesses.

The negative health effects of trans fatty acids from hydrogenated fats on plasma lipid profile have been well documented. However, the mechanisms responsible for these changes remain to be elucidated. Hence the overall objective of the thesis was to examine the effect of consuming different forms of hydrogenated fats on cholesterol and triglyceride metabolism, specifically fractional and absolute synthesis rates of free (FSR-FC and ASR-FC) and esterified (FSR-CE, AER and ER) cholesterol, and the functioning of the ASP pathway. In addition, validation of the newer deuterium incorporation (DI) method for measurement of endogenous cholesterol biosynthesis against the cholesterol precursor assessment approach was also performed. Fourteen moderately hypercholesterolemic (LDL-C ≥ 130 mg.dl-1) postmenopausal women (65--71yrs) participated in this study. Subjects consumed, in random order, each of 6 diets for 5 week periods, separated by washout periods ranging from 2 to 4 weeks in duration. The experimental diets included a baseline (BL) diet (39% kcal fat) and 5 reduced fat diets (30% kcal) where 2/3rd of the fat was either soybean oil (SO), low trans squeeze (SQM), medium trans tub (TM), high trans stick (SM) margarines, or butter (BT). Results obtained from the series of analyses performed demonstrate that: (i) the DI method and levels of some cholesterol precursors correspond as methods for the study of in vivo cholesterol biosynthesis in humans; (ii) elevations in endogenous cholesterol synthesis (FSR-FC and ASR-FC) are not responsible for the increase in circulating cholesterol levels seen after consumption of the high trans SM, and high SFA rich BT and BL diets; (iii) suppression of cholesterol esterification rates on the SM diet may account for the decreased HDL-C levels observed on this diet and finally; (iv) dysfunction of the ASP pathway, with lower ASP and higher FFA levels could be responsible for the higher secretion of hepatic B 100 particles. In conc

No description available.

Docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6) are deposited in large amounts in membrane phospholipids of the developing central nervous system (CNS). High levels of 22:6n-3 are found in synaptic terminals and retina, and are important for normal visual development and function. 20:4n-6 and22:6n-3 are supplied in human milk. In contrast, infants fed formula rely completely on endogenous synthesis of 20:4n-6 and 22:6n-3 from linoleic (18:2n-6) and a-linolenic (18:3n-3) acid, respectively. Levels of 22:6n-3 in the blood lipids of infants fed formula are lower than in infants fed human milk. Concern over the supply of 22:6n-3 led to clinical trials in which premature infants were fed formulas containing fish oils as a source of 22:6n-3. Piglets, which have a similar lipid metabolism and perinatal timing of the brain growth spurt to humans, have a lower percentage of 22:6n-3 in blood, liver and CNS tissues when fed formula with 30% of fatty acids as18:2n-6 and 0.8% 18:3n-3, compared to sow milk. It was hypothesized that the low blood and tissue 22:6n-3 in formula-fed piglets was due to inappropriate quantities and/or ratios of dietary 18:2n-6 and 18:3n-3 limiting the synthesis of 22:6n-3. Thus, the main objectives of this thesis were to determine. (1) if 22:6n-3 is an essential dietary nutrient for the term gestation piglet, (2) if appropriate quantities and ratios of 18:2n-6 and 18:3n-3 in formula will support CNS membrane accretion of 20:4n-6 and 22:6n-3, comparable to piglets fed varying amounts of 22:6n-3 in natural milk, and (3) if lower blood phospholipid 22:6n-3 consistently reflects reduced 22:6n-3 in the CNS. Initial studies (Experiment I) showed that formula with 4% 18:3n-3 supported a similar percentage of22:6n-3 in piglet liver and CNS membrane lipids to sow milk, but was associated with lower brain weight. Deposition of 22:6n-3 in brain was influenced by the formula 18:3n-3 content. The 18:2n-6:18:3n-3 ratio (22:1and 37:1) seemed to be important, however, when formulas contained 1% 18:3n-3. Low levels of fish oil in formula, similar to those used in clinical trials, were effective in supplying 22:6n-3 to the developing piglet brain (Experiment II). The efficacy of 18:3n-3 in supporting the deposition of 22:6n-3 in the brain was estimated to be at least 20% that of dietary 20:5n-3 plus 22:6n-3. With increasing dietary fish oil, however, levels of eicosapentaenoic acid (20:5n-3) increased and 20:4n-6decreased in plasma, liver and retina, but not brain (Experiment III). This suggests regulatory mechanisms may exist to maintain relatively constant levels of 20:4n-6 and 20:5n-3 in brain. Milk 22:6n-3 varies with maternal intake of 22:6n-3. The effect of milk 22:6n-3 content was studied in piglets fed milk with 0.1% or 1.5% 22:6n-3 obtained from sows fed usual pig diets containing vegetable fats without or with fish oil, respectively (Experiment IV). Consumption of 1.5 vs 0.1% 22:6n-3 from sow milk resulted in 300% higher 22:6n-3 in liver and blood phospholipids and 11% higher 22:6n-3 in cerebrum of nursing piglets. Despite similar milk 20:4n-6, the % 20:4n-6 in tissues other than the brain was lower in piglets fed high22:6n-3 sow milk. Thus, high intakes of n-3 fatty acids decrease 20:4n-6 in piglet liver and blood lipids. The blood phospholipid % 22:6n-3 in piglets fed formulas containing 18:2n-6 and 18:3n-3 but not their long-chain derivatives, was lower than in piglets fed 22:6n-3 in natural milk, consistent with published findings in formula-fed infants. However, in contrast to circulating lipids, formulas with 4% 18:3n-3 maintained similar levels of 22:6n-3in the piglet CNS compared to milk. These studies show that blood phospholipid 22:6n-3 and 20:4n-6 are not specific indices of effects in CNS lipids. This thesis has shown (1) 22:6n-3 is not essential in the diet of the term piglet, if adequate 18:3n-3 is given, (2) fish oils are an effective source of 22:6n-3 for deposition in the developing brain, (3) high dietary n-3fatty acids interfere with 20:4n-6 metabolism, and (4) blood lipid 20:4n-6 and 22:6n-3 do not accurately reflect CNS fatty acids.