(hCysLT2R) based on a β-galactosidase complementation system. The EC50 values for LTC4 and LTD4 are consistent with previous literature values determined based on radio-labelling affinity assays for hCysLT2R. Activity of a synthetic analogue of LTC4, N-Methyl LTC4 (NMLTC4), gave an EC50 value of 8.5 nM. Also described in this thesis, is the synthesis of a cysLT2R selective antagonist, 3-{[(3- carboxycyclohexyl)amino]carbonyl}-4-{3-{4-(4-phenoxybutoxy)phenyl}- propoxy}benzoic acid. Through the in vitro assay system, this selective antagonist showed a dose-dependent inhibition (IC50 value of 86 nM) when CysLT2R was stimulated with 30 nM of LTD4. Both the selective agonist (NMLTC4) as well as the selective cysLT2R antagonist were also tested in vivo in a localized vascular ear inflammation assay. Results show NMLTC4 is able to promote vascular leakage through stimulation of cysLT2R, and this extravasation can be significantly attenuated by the cysLT2R antagonist. / Thesis (Master, Biochemistry) -- Queen's University, 2011-06-29 22:31:03.11

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Submerged cultures of Aspergillus flavus and Aspergillus niger were grown in a medium containing avidin, a substance which serves as an inhibitor of the conversion of acetate to malonate. Control cultures were grown without the addition of avidin.Analysis of the fatty acids produced by cultures grown in a medium containing avidin gave an increase in C16 fatty acids and a decrease in C18 fatty acids, suggesting that malonate plays an important role in the elongation of long chain fatty acids in these organisms. This effect was observed for five to fifteen hours.Evidence of the conversion of palmitate to stearate to oleate to linoleate was presented. That the conversion of oleate to linoleate involves a desaturase which is highly specific is suggested by the observation that, although there are a number of monoenoic acids present in these organisms, the only dienoic acid found was linoleic acid.

The work detailed in this thesis is mainly concerned with the carbonylation of fatty acid derivatives, using carbon monoxide atmospheres in the presence of a transition metal. The two systems under study were the conversion of oleonitrile to a cyano-ester using a cobalt catalyst, and formation of isocyanates from amines in the presence of a palladium salt. The objectives of each study were: (i) to assess the influence of physical conditions on the reaction (i.e. pressure, time and temperature);(ii) to examine the effect of various additives on the system (i.e. phosphines, iodides and bases); and (iii) to identify any catalytic species observed under reaction conditions.Three major techniques were employed in each study: autoclave reactions, high pressure infra-red studies and atmospheric pressure nitrogen-line chemistry. The proposed underlying reaction mechanism was examined critically for each system, comparisons being drawn between the postulated catalytic species and those actually observed by high pressure infra-red spectroscopy. The chemical properties of the proposed catalytic intermediates are discussed in relation to the effect of co-reactants on the system. In the case of the carbonylation of oleonitrile the high pressure infra-red studies are consistent with the proposed reaction mechanism based upon the acyl complex RCOCo(CO)(_4). Although the infra-red study on the carbonylation of amines did not reveal any definite information, the results of other experiments were found to be consistent with a system based upon a carboxamido species L(_2)Pd(Cl)(CONRR').

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Dietary docosahexaenoic acid (22:6n-3, DHA) has been shown to exert beneficial effects on coronary heart disease including the prevention of ischemia reperfusion injury. The ability to acutely infuse DHA to the heart to prevent ischemia reperfusion injury is a potentialy valuable tool in planned surgery where reperfusion and/or ischemia will take place including coronary artery bypass surgery and angioplasty. In the present study, hearts from chow-fed (AIN-93M) Sprague Dawley rats (male) 9-12 weeks of age were isolated and artificially perfused. The protocol included: 30 min stabilization period, 30 min global no flow ischemia, 15 min fatty acid infusion with reperfusion, and 75 min reperfusion in the absence of fatty acids. The fatty acid infusions included 10, 20, 40, 60, 80, 100 or 120 µM of either palmitate or DHA complexed to 3% essentially fatty acid free bovine serum albumin as well as a vehicle control. Heart functional data was recorded continuously and total heart infarct volume was determined after staining with triphenyltetrazolium chloride. DHA at 10µM significantly reduced the infarction area at the end of the reperfusion period compared to that observed in the10µM of palmitate and vehicle control conditions. Infarction areas after infusions with DHA or palmitate were similar to controls after 20-60 µM infusions and greater than controls after 80-120 µM infusions, except for the 100 µM palmitate conditions which were similar to the low and high doses. In this model of infusion, 120 µM of fatty acid was the maximum amount of DHA tolerated, as several hearts went into fibrillation and did not recover and failed to complete the reperfusion at concentrations greater than 120 µM of DHA. DHA and palmitate also exerted dose dependent effects on functional parameters. In summary, infusion of DHA and palmitate cause dose dependent effects on heart function.

Monounsaturated fatty acids (MUFA) have been viewed as either beneficial or neutral with respect to health; however, recent evidence suggests that MUFA may be associated with obesity and cardiovascular disease. Sex differences in MUFA composition have been reported in both rats and humans that may be mediated by sex hormones. Therefore, differences in fatty acid and enzyme expression were examined in male and female rats. HepG2 cells treated with 17β-estradiol, progesterone and testosterone (0, 10, 30 and 100 nM), and ovariectomized female rats with hormone implants (sham, no treatment, estradiol, progesterone, and estradiol plus progesterone) examined the role of sex hormones in MUFA metabolism. MUFA concentrations were determined by gas chromatography. The mRNA and protein expression of stearoyl-CoA desaturase and elongase 6, key enzymes involved in MUFA biosynthesis, were measured by real-time PCR and immunoblotting, respectively. Elongase 6 protein expression was higher in females as compared with males, increased with estradiol and progesterone treatment of HepG2 cells, and was higher in ovariectomized rats treated with estradiol. Elongase 6 expression was also decreased in HepG2 cells treated with testosterone. In contrast, the expression of stearoyl-CoA desaturase did not appear to be associated with sex or sex hormones in HepG2 cells, although ovariectomized rats treated with estradiol plus progesterone had increased stearoyl-CoA desaturase protein levels compared with sham controls. Sex differences and differences after hormonal treatments were observed in the fatty acid concentrations of MUFA and immediate MUFA substrates including 16:0, 16:1n-7, 18:0, 18:1n-7 and 18:1n-9. These differences in MUFA are consistent with the changes in elongase 6 expression. The effect of sex and hormone status on elongase 6 has not been previously examined, but these results suggest elongase 6 is an important factor in determining MUFA composition. These sex and hormonal differences in MUFA composition may contribute to sex differences in obesity and cardiovascular disease. Further work examining other factors involved in MUFA composition including oxidation, synthesis of complex lipids and the effect of diet is required.

Two methods for the estimation of non-esterified fatty acids in plasma were evaluated. An attempt was made to obtain a reliable micro-technique to estimate these acids in blood fractions. The titrimetric method of Trout et al. was satisfactory in the analyses of pure fatty acids and fatty acids added to serum. In the analyses of palmitic and stearic acid solutions, the mean values ranged from 95 to 115 percent of actual values. The mean recovery of palmitic acid added to a serum was 110 percent. The reproductibility of the method was fair in replicate analyses of three standard sera on two to four different days. The mean values determined for Serum A were 645, 653, 641, and 605; for Serum B, 587 and 587; and for Serum C, 565, 571 and 533 μEq per liter of serum. The colorimetric micro-method of Mendelsohn was also satisfactory in the analyses of pure fatty acids, fatty acids added to a serum, and a fatty acid as a component of standard lipid mixtures. In the analyses of palmitic, stearic, and oleic acid solutions, the mean values ranged from 97 to 118 percent of actual values. The recoveries of palmitic and oleic acids added to a serum ranged from 81 to 111 percent in four different experiments. In the determination of oleic acid from mixtures of several lipids, the recovery ranged from 95 to 107 percent. The reproductibility of the method was also fair in replicate analyses of a standard serum on 11 different days. The individual determinations ranged from 768 to 1046 μEq per liter of serum with a mean of 917. The mean values of 11 different experiments ranged from 830 to 1013 μEq per liter of serum. A comparison of methods was made by analyzing four standard sera in replicate by both methods. The values of the non-esterified fatty acids by the method of Mendelsohn were approximately twice those by the method of Trout et al. The difference was attributed to the degrees of extraction of non-esterified fatty acids and/or other acids from the serum by the two methods. The concentrations of the non-esterified fatty acids in the sera and the erythrocytes of 16 subjects of both sexes, ranging in age from 23 to 66 years, were determined by an adaptation of the colorimetric method of Mendelsohn. The concentrations in the sera ranged from 380 to 1057 μEq per liter with a mean of 800. The concentrations in the erythrocytes were consistently higher than those in the serum for all the subjects and ranged from 552 to 1919 μEq per liter with a mean of 1362. / Graduation date: 1965