Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "May 2002." Typescript. Includes bibliographical references. Also available on the Internet.

This study was conducted to test the silver spoon hypothesis that earlylife nutritional conditions impact development, performance and fitness of the birdsgreat tit (Parus major) nestlings. We investigated whether fatty acid affects immunityand metabolism during the altricial period by examining the expressions of geneTLR4 (immunity related) and COX 2 (metabolism related) against a dietarymanipulation on great tit nestlings in urban vs. rural environments. The resultssuggested that arachidonic acid had no significant effect on TLR4 expression, but atendency to induce immune response, regardless of urban or rural conditions. Thestrength of immune response was however negatively correlated with laying date. Theurban great tit nestlings had a higher COX 2 gene expression than rural ones, andarachidonic acid suppressed COX 2.Thus no strong support to the hypothesis was found for the studied great titpopulations. It showed, however, i) there is a tendency of increasing immune responsewith extra fatty acid in the diet, and ii) arachidonic acid suppress metabolism. Fattyacid involved in a multiple physiological processes and this complex need to beelaborated in future studies.

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.

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Most embryonal mortality occurs during early embryonic development. Two experiments were designed to study aspects of early embryonic development. Experiment 1 was to determine if early ovine embryos were capable of metabolizing arachidonic acid. Cyclic ewes were estrous synchronized with 6⍺-methyl-17β-hydroxy progesterone acetate (MPA) pessaries, superovulated with follicle stimulating hormone (FSH) and bred artificially. Embryos were collected on d 4, 8, 10, 12 or 14 of pregnancy and incubated with 1 μCi of [¹⁴C] arachidonic acid in an atmosphere of 5% CO₂, 45% O₂ and 50% N at 37°C for 24 h. Embryos from all days of pregnancy metabolized arachidonic acid to a number of compounds. Embryos produced primarily an unidentified polar compound, 6-keto-prostaglandin F₁⍺ (6-keto-PGF₁⍺), prostaglandin F₂⍺ (PGF₂⍺), prostaglandin E₂ (PGE₂), 13,14-dihydro-15-keto prostaglandin F₂⍺ (PGFM), prostaglandin B₂ (PGB₂) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Experiment 2 was to determine whether prostaglandins have a role in embryo hatching from the zona pellucida. Ewes were superovulated and bred artificially, and embryos were collected on d 7 of pregnancy. Embryos were incubated with ethanol (control), indomethacin, PGE₂ or indomethacin and PGE₂ in an atmosphere of 5% CO₂ and 95% air at 37°C for 24 h. Indomethacin appeared to decrease embryo hatching rate (indomethacin, 34.5% vs control, 46.4%). Prostaglandin E₂ appeared to increase embryo hatching rate (PGE₂, 60.0% vs. control, 46.4%). However, hatching rates for indomethacin and PGE₂ treatment groups were not different from control (P > .05). When compared to any group with indomethacin treatment, PGE₂ increased (P < .05) embryo hatching rate. The results of this study indicated that early ovine embryos can convert arachidonic acid to various compounds in vitro. Although not conclusive, indomethacin may decrease and PGE₂ may increase embryo hatching rate. Therefore, embryo-produced prostaglandins may be involved in hatching of sheep embryos from the zona pellucida. / Master of Science

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Includes bibliographical references. Also available on the Internet.

[Truncated abstract] Arachidonic acid is oxidised in vivo by non-enzymatic (free radical) or enzymatic pathways (cyclooxygenase, lipoxygenase, and cytochrome P450) to form a range of biologically active eicosanoids. Specifically, arachidonic acid is metabolised by cytochrome P450 -hydroxylase to produce vasoactive 20-hydroxyeicosatetraenoic acid (20-HETE), and by 5-lipoxygenase to produce proinflammatory leukotriene B4 (LTB4), which can further be metabolised by -hydroxylase to from 20-OH-LTB4 and 20-COOH-LTB4. F2-Isoprostanes (F2-IsoPs) are produced through free radical attack on arachidonic acid and have been recognised as the most reliable markers of lipid peroxidation in vivo. The metabolic syndrome (MetS) is characterised by abdominal obesity, hypertension, insulin resistance, glucose intolerance, and dyslipidemia. It is associated with low-grade inflammation and oxidative stress and an increased risk of developing cardiovascular diseases. Dietary weight loss is strongly recommended for the management of the MetS and can potentially minimise the risk of cardiovascular diseases and diabetes in individuals with the MetS. Little is known regarding the role of these arachidonic acid metabolites in the MetS and the effect of weight loss on their metabolism. Chapter three comprised of three in vitro studies aimed to examine 20-HETE synthesis in human blood cells. 20-HETE acts as a second messenger for vasoconstrictor actions of angiotensin II (Ang II) and endothelin-1 (ET-1) in renal and mesenteric beds. Human neutrophils and platelets are integral to the inflammatory process. ... Production of LTB4 and 20-OH-LTB4 was significantly lower compared with controls (P<0.005) and remained so after adjustment for neutrophil count (P<0.05).The weight loss intervention resulted in a 4.6kg reduction in body weight and a 6.6cm decrease in waist circumference and a significant increase in LTB4 and 20-OH- LTB4 in the weight loss group. Chapter Five continued to investigate the role of other arachidonic acid metabolites, 20-HETE and F2-IsoPs in the MetS and the effect of weight loss. In the case-control study (Human study 1), plasma and urinary 20-HETE and F2-IsoPs were significantly elevated in the MetS group, but no significant difference was found in stimulated-neutrophil 20-HETE. A significant gender x group interaction was observed in that women with the MetS had higher urinary 20-HETE and F2-IsoPs compared to controls (P<0.0001). In a randomised controlled trial (Human study 2), relative to the weight- maintenance group, a 4.6 kg loss in weight resulted in a 2 mmHg fall in blood pressure but did not alter the production of 20-HETE or F2-IsoPs. No significant differences were shown in 20-HETE released from stimulated-neutrophils before and after weight loss. 20-HETE and oxidative stress may be important mediators of cardiovascular disease risk in the MetS. Although a 4% reduction in body weight reduced BP, there were no changes in plasma or urinary 20-HETE or F2-IsoPs. In summary, in vitro studies show that human neutrophils and platelets can produce 20-HETE in response to Ang II and ET-1, and human studies demonstrate that the presence of MetS has a significant impact on arachidonic acid metabolism and effective weight loss can restore leukocyte synthesis of LTB4.

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<p>Prostaglandin (PG) E₂ is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE₂ has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE₂ is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE₂ synthesising enzymes are not completely established.</p><p>PGE₂ was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA₂a (cPLA₂a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE₂. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH₂. The conversion of PGH₂ to the final product PGE₂ was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA₂α was markedly advantageous for the formation of PGE₂.</p><p>Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE₂ in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA₂a.</p>