It is well established that a cardiomyopathy independent of atherosclerosis develops in both humans and animals with diabetes mellitus. The etiology of diabetic cardiomyopathy is very complex involving many different processes, one of which may be the increased fatty acid utilization, and/or the concomitant decrease in glucose utilization, by the diabetic heart. We compared control and 6-week streptozotocin(STZ)-induced diabetic isolated working rat hearts and were able to demonstrate cardiac dysfunction in the diabetic as assessed by depressed heart rate (HR), heart rate peak systolic pressure product (HR X PSP), left ventricular developed pressure (LVDP), rate of pressure rise (+dP/dt) and rate of pressure decline (-dP/dt). Paralleling depressed cardiac function in the diabetic were hyperglycemia, hyperlipidemia and decreased body weight gain as compared to age-matched controls. The addition of free fatty acids, in the form of 1.2 mM palmitate, to the isolated working heart perfusate had no effect on either control or diabetic heart function, with the exception of a depressive effect on +dP/dt of diabetic hearts and -dP/dt of control hearts. But, diabetic hearts perfused with palmitate-containing perfusate plus the glucose oxidation stimulator dichloroacetate (DCA) showed a marked improvement in function. Heart rate, HR X PSP, LVDP and +/-dP/dt were all restored to control heart values in diabetic hearts perfused in the presence of DCA. Creatine phosphate and adenosine 5'-triphosphate (ATP) levels were similar under all perfusion conditions, therefore eliminating energy stores as the limiting factor in heart function. Results indicate that DCA-induced stimulation of glucose oxidation acutely reversed diabetic cardiac function depression. Therefore, depression of glucose oxidation in the diabetic heart may be contributing to diabetic cardiomyopathy. / Pharmaceutical Sciences, Faculty of / Graduate

The potential use of four radioiodinated fatty acids as myocardial imaging agents were evaluated. Preliminary distribution studies revealed that the terminal labeled fatty acids demonstrated higher myocardial uptake. Thus, 10-Iodocapric acid (10-iododecanoic) and 12-Iodolauric acid were subjected to further investigation. Comprehensive tissue distribution studies involving both radioiodinated fatty acids in mice indicated that the highest accumulation of the total injected activity occurred in the muscle and the blood. Other organs investigated included the heart, liver, lung, kidneys, spleen, stomach, intestines, bone and adrenals. The heart exhibited the highest concentration of the radioiodinated fatty acids for the relative accumulation of activity per unit weight. Erom the tissue distribution studies, the optimum scanning time was found to be immediately following injection of the radiopharmaceutical. Toxicity studies were performed in mice after intravenous administration of 10-Bromocapric acid and 12-Bromolauric acid. The LD₅₀ of Sodium Bromolaurate in 10% Human Serum Albumin was found to be 210 mg/kg (194 mg/kg - 228 mg/kg). The stability problem encountered with 10-Bromocapric acid necessitated the use of a different solvent system. The LD₅₀ obtained after intravenous injection was found to be 86.1 mg/kg (83.0 mg/kg - 89.3 mg/kg). However, this observed toxicity may not necessarily reflect the toxicity of the Bromocapric acid solely. Whole body excretion studies were performed in mice and revealed a triexponential excretion curve. For 10-Iodocapric acid, the effective half-lives were .90 hours (36.7%), 3.91 hours (61.6%) and 74.9 hours (14.5%). For 12-Iodolauric acid, the effective half-lives were 1.67 hours (46.6%), 7.68 hours (38.4%), and 71.6 hours (17.8%). For both 10-Iodocapric acid and 12-Iodolauric acid, the first as well as the second component of the excretion curve presumably represented a decrease in the whole body activity due mainly to urinary excretion. The third component appeared to represent activity which was tightly bound and slowly released. The third component presumably represented elimination by fecal excretion. The excretion of the injected activity was primarily in the urine, although some activity was recovered in the feces. For 10-Iodocapric acid, 82.4% of the injected activity had been recovered in the urine within the first 24 hours and 8.88% had been recovered in the feces. For 12-Iodolauric acid, 78.9% of the injected dose was recovered in the urine at 24 hours and 9.4% in the feces. From the urine results, the effective half-life of the radio-iodinated fatty acids in the kidneys was found to be 4.8 hours. Myocardial scans were done on rabbits using ¹³¹I-capric acid, ¹³¹I-lauric acid, NaI-131 (6% Human Serum Albumin), and Thallium-201 at specified time intervals after injection. Iodine-123, a radionuclide possessing more favorable imaging properties, was not readily available due to production problems at the time of scanning. The mean absorbed dose to the whole body, the liver, the kidneys, the muscle, and the heart were computed based on the results from the distribution and excretion studies. The dosimetry calculations were done using Iodine-123 as the radionuclide. For ¹³¹I-capric acid, the radiation doses were calculated as 34.76 mrads/2 mCi for the whole body, 136.3 mrads/2 mCi for the kidneys, 86.6 mrads/2 mCi for the liver, 38.5 mrads/2 mCi for the muscle, and 25.89 mrads/2 mCi for the heart. For ¹³¹I-lauric acid, the radiation doses were 41.73 mrads/2 mCi for the whole body, 199.8 mrads/2 mCi for the kidneys, 185.9 mrads/2 mCi for the liver, 52.07 mrads/2 mCi for the muscle, and 46.39 mrads/2 mCi for the heart. / Pharmaceutical Sciences, Faculty of / Unknown

Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild type Arabidopsis plants regarding their sensitivity to low temperature, and the size and height of the plants.

Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active and inactive AtFAAH overexpressors displayed hypersensitivity to ABA, suggesting a function of the enzyme independent of its catalytic activity toward NAE substrates. Yeast two-hybrid screening identified Arg/Ser-rich zinc knuckle-containing protein as a candidate protein that physically and domain-specifically interacts with AtFAAH and its T-DNA knock-out Arabidopsis was hypersensitive to ABA to a degree similar to AtFAAH overexpressors. Taken together, AtFAAH appears to have a bifurcating function, via NAE hydrolysis and protein-protein interaction, to control Arabidopsis growth and interaction with phytohormone signaling pathways. These studies help to functionally define the group of enzymes that metabolize NAEs in plants, and further will expand the knowledge-base of lipid metabolism and signaling for manipulation of various physiological processes important to plant growth and responses to environmental stress.

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.

No description available.

Tree peony (Paeonia section Moutan DC.) species are woody oil crops with high unsaturated fatty acid content, including α-linolenic acid (ALA/18:3; >40% of the total fatty acid). Comparative transcriptome analyses were carried out to uncover the underlying mechanisms responsible for high and low ALA content in the developing seeds of P. rockii and P. lutea, respectively. Expression analysis of acyl lipid metabolism genes revealed upregulation of select genes involved in plastidial fatty acid synthesis, acyl editing, desaturation, and triacylglycerol assembly in seeds of P. rockiirelative to P. lutea. Also, in association with ALA content in seeds, transcript levels for fatty acid desaturases (SAD, FAD2, and FAD3), which encode enzymes necessary for polyunsaturated fatty acid synthesis, were higher in P. rockii compared to P. lutea. Furthermore, the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and ALA content, respectively, and modulated the final ratio 18:2/18:3 in the seed oil. In conclusion, we identified the key steps and validated the necessary desaturases that contribute to efficient ALA synthesis in a woody oil crop. Together, these results will aid to increase essential fatty acid content in seeds of tree peonies and other crops of agronomic interest.

The overall purpose of this thesis is to understand the impact of management on commercial egg layers, whether that be environmental-related, health-related, or other possible stressors within the pullet and post-peak phases. Furthermore, the study seeks to examine what effects to performance and production these impacts may induce within a commercial layer in differing phases, specifically the pullet and post-peak phases. Chapter two explores into coccidiosis within the pullet phase, which induced by a commercial vaccine, can provide stress to a pullet, lowering protection against infection, and seriously compromising its growth and development into peak lay. Chapter three explores into the post-peak phase, a transition from a caged system of production to cage-free system of production was selected. incorporating multiple differing environmental stimuli that can induce stress. If commercial layers prove to possess the capabilities to adapt to these impacts in multiple phases, the possibility of extended production is possible.

Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants. The organism is unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP). The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants. The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and β-ketoacyl-ACP synthase I. The effects of inhibitors on the three different enzymes were noted. All carbon-carbon bond formation was inhibited by cerulenin. Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents. Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA. There was no evidence for a β-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria. The roles of the acetyl-CoA-ACP transacylase and β-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena. Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and β-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights.

Bisphenols are used in the process of polymerization of polycarbonate plastics and epoxy resins. Bisphenols can easily migrate out of plastic products and enter the gastrointestinal system. By increasing colonic inflammation in mice, disrupting the intestinal bacterial community structure and altering the microbial membrane transport system in zebrafish, bisphenols seem to interfere with the gut microbiome. The highly abundant human commensal bacterium Bacteroides thetaiotaomicron was exposed to bisphenols (Bisphenol A (BPA), Bisphenol F (BPF), Bisphenol S (BPS)), to examine the mode of action, in particular of BPF. All chemicals caused a concentration-dependent growth inhibition and the half-maximal effective concentration (EC50) corresponded to their individual logP values, a measure of their hydrophobicity. B. thetaiotaomicron exposed to BPF decreased membrane fluidity with increasing BPF concentrations. Physiological changes including an increase of acetate concentrations were observed. On the proteome level, a higher abundance of several ATP synthase subunits and multidrug efflux pumps suggested an increased energy demand for adaptive mechanisms after BPF exposure. Defense mechanisms were also implicated by a pathway analysis that identified a higher abundance of members of resistance pathways/strategies to cope with xenobiotics (i.e., antibiotics). Here, we present further insights into the mode of action of bisphenols in a human commensal gut bacterium regarding growth inhibition, and the physiological and functional state of the cell. These results, combined with microbiota-directed effects, could lead to a better understanding of host health disturbances and disease development based on xenobiotic uptake.