No description available.

Epidemiological studies have shown that cholesterol is a major risk factor for the development of atherosclerosis. Since the atherosclerotic plaque develops over a long period interventions early in life may be of some benefit. In addition, it has been shown that the enzymes involved in cholesterol metabolism can be manipulated in early life. Therefore, studies of the developmental patterns of the key enzymes in cholesterol metabolism are of great importance. Acyl coenzyme A: cholesterol acyltransferase (ACAT) is the primary enzyme which catalyzes the conversion of free cholesterol to cholesterol esters in cells. A better understanding of the role and control of ACAT during development is needed in order to trace the possible causes in early life that lead to atherosclerosis in the adult. This research focused on the developmental pattern of ACAT in the rat liver, intestine, brown and white adipose tissue (BAT and WAT, respectively) and aorta. Age specific changes were observed in the rat liver, intestine and BAT. The rat liver and intestine possess significant amounts of ACAT activity throughout development and there appears to be marked variations in activity during this time. The rat BAT and WAT appear to be devoid of ACAT activity throughout development with the exception of adult BAT. Due to the small amount of the aortic tissue samples and/or the insensitivity of the assay, no definite conclusions could be made from this aortic study. In searching for factors that might control the ACAT enzyme the immediate effects of short-term manipulation of diet on the activity of ACAT were studied. The rats were all weaned early on day 18 to one of the following diets: Purina Rat Chow, high carbohydrate (HG) , high fat (HF) , or 2% cholesterol. The HF was the only diet that consistently increased hepatic ACAT activity in all the age groups. The cholesterol diets significantly increased the activity of ACAT in the 22 and 25 day old rats. The HG diet increased the activity of ACAT in the 22, 25, and 30 day old rats. No significant differences were observed between the adult control and HG diet groups. Feeding rats a HF or HG diet precipitated a dramatic drop in intestinal ACAT activity in the 22 day old animals. These effects were not observed in the older animals. The high cholesterol diet had no significant effect on the intestinal enzyme's activity in 22 day old rats. There was no significant change in the BAT and WAT ACAT activity with the experimental diets with the exception that all the experimental diets decreased ACAT activity in the adult BAT. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

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The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.

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The infestation of twelve selected clones of alfalfa by the alfalfa seed chalcid was measured. The free sterol content of the stems, leaves, f lowers and pods of these clones was determined. A check was made to see if there was a correlation between the sterol content and the chalcid infestation. Differences were found among t he clones in the sterol content for each plant part. There were also significant differences in percent chalcid infestation in the 12 clones. Duncan ' s New Multiple Range Test pointed out which clones differed Significantly from the others. Using linear regression analysis, the correlation coefficient of the comparison between the sterol content and the chalcid infestation was obtained. There was a signtflcant negative correlation between the sterol content of the flower and the percent chalcid infestation. No significant correlation was found between t he sterol content of the stems, leaves or pods and the percent chalcid infestation.

Reduced bioavailability in some formulations of phytosterols accounts for the variable results observed in LDL- C- lowering efficacy among trials. This study examined the effects of a water-dispersible formulation of free phytosterols (WD-PS) versus phytosterol esters (PS-esters) on plasma lipid and fat soluble vitamins concentrations in hypercholesterolemic individuals. Using a double-blind, randomized, crossover study, 47 hypercholesterolemics were provided for 4 wk: WD-PS-enriched yogurt (2g/d), PS-esters-enriched yogurt (2g/d), or yogurt alone (placebo), in a random order. Each study phase was separated by 4 wk washout intervals. Supplementation of WD-PS or PS-esters similarly decreased serum TC (7.7% and 6.3%, respectively) and LDL-C levels (11.7% and 11.6%, respectively, p<0.001). The ratio of TC/ HDL-C decreased for WD-PS (10.6%, p<0.05) but not for PS-esters. Moreover, WD-PS reduced serum TG (13.9%, p<0.05) as compared to PS-esters (0.6%). The results of the current study confirm the importance of the formulation of phytosterols in their bioavailability and efficacy.

Sterols have been identified as major components of membrane lipids that are part of specialized membrane domains necessary for organizing events such as polar protein targeting and signal transduction in plants, fungi and animals. However a common modification of sterols is the addition of sugar moieties via glycosylation abundantly found in plants. An exact physiological role for such diversification of sterols in plants is still unknown. Using reverse genetics and transcriptomics we show that UDP-glucose: sterol glucosyltransferase encoded by UGT80B1 is necessary for correct epidermal patterning in Arabidopsis root. Patterning of hair cells (trichoblasts) and non-hair cells (atrichoblasts) in the epidermis of the Arabidopsis root involves signaling through SCRAMBLED (SCM), a plasma membrane localized LRR-RL kinase. Feedback regulation via the transcriptional regulatory complex containing R2R3-MYB transcription factor WEREWOLF (WER) represses SCM and activates the homeodomain-leucine-zipper protein GLABRA2 (GL2) in atrichoblasts. Evidence suggests symplastic connections between cells, known as plasmodesmata, establish passage ways for single-repeat R3-MYB transcription factors to activate SCM expression in trichoblasts. Mutations in UGT80B1 cause atypical localization patterns of GL2, WER, and SCM in the root epidermis. The ugt80B1 formed fewer trichoblasts in comparison to wild-type. A translational fusion of UGT80B1 to GFP localizes to the ER, plasma membrane and to sites that appear to be plasmodesmata-associated desmotubules. Ultrastructural analysis revealed abnormalities in plasmodesmata formation and morphology in ugt80B1 mutants. Steryl glucoside profiling indicated deficiencies in specific glycosylated sterol compounds in roots. This study identifies UGT80B1 as a novel membrane component that is critical for plasmodesmata morphogenesis and cell-fate determination in the root epidermis. A model is proposed in which UGT80B1 activity provides spatially discreet sterol and steryl glucoside architecture within the plasma membrane to anchor the SCM receptor and within plasmosdesmata to facilitate intercellular movement of R3-MYB regulatory proteins underlying proper differentiation of trichoblasts versus atrichoblasts. Moreover, evidence from reverse genetics, proteomics and live cell imaging point to a actin dependent localization of UGT80B1 at the vesicle rich zone of root hair tip. This localization actively supports root hair elongation via tip growth, possibly by membrane modifications required for vesicle trafficking.

Reduced bioavailability in some formulations of phytosterols accounts for the variable results observed in LDL- C- lowering efficacy among trials. This study examined the effects of a water-dispersible formulation of free phytosterols (WD-PS) versus phytosterol esters (PS-esters) on plasma lipid and fat soluble vitamins concentrations in hypercholesterolemic individuals. Using a double-blind, randomized, crossover study, 47 hypercholesterolemics were provided for 4 wk: WD-PS-enriched yogurt (2g/d), PS-esters-enriched yogurt (2g/d), or yogurt alone (placebo), in a random order. Each study phase was separated by 4 wk washout intervals. Supplementation of WD-PS or PS-esters similarly decreased serum TC (7.7% and 6.3%, respectively) and LDL-C levels (11.7% and 11.6%, respectively, p<0.001). The ratio of TC/ HDL-C decreased for WD-PS (10.6%, p<0.05) but not for PS-esters. Moreover, WD-PS reduced serum TG (13.9%, p<0.05) as compared to PS-esters (0.6%). The results of the current study confirm the importance of the formulation of phytosterols in their bioavailability and efficacy.

No description available.