Browsing by Subject "Cholesterol"
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Item A comprehensive study of cholesterol in biomembranes using computer simulations(2011-05) Dai, Jian; Huang, Juyang; Park, Soyeun; Sanati, Mahdi; Khare, RajeshSeveral methods were applied to study the effects of cholesterol on multi-component lipid bilayers. The goal is to investigate the validity of the "Umbrella Effect" via simulations and experiments. The Umbrella Model is a hypothesis proposed based on previous structural and thermodynamical studies of lipid membranes containing cholesterol. DPPC is the most widely studied phospholipid in the simulation community. It has a large polar headgroup (which consists of a positively charged choline group and a negatively charged phosphate group), a glycerol group and two long saturated hydrocarbon chains. On the other hand, cholesterol has a relatively large carbon-ring body, compared to its small hydrophilic hydroxyl group. When a binary mixture of DPPC/cholesterol is placed in an aqueous environment, it has been suggested that cholesterol is always trying to find a “shield” to protect it from extensive contact with water, and this shield is most likely to be provided by the headgroups of phospholipids. This hypothesis is termed the “Umbrella Model”. Monte Carlo (MC) simulations will be used to study the phase transitions of multi-component lipid mixtures and molecular dynamics (MD) simulations will be used to test the Umbrella Model in direct and indirect ways, and to interpret the experimental data. Melittin is the most studied antimicrobial peptide, it can cause cell death by damaging cell membranes. Melittin interacts differently with various membrane components, such as cholesterol and negatively-charged phospholipid, both of which have been shown to reduce melittin's lytic effect against the membrane. Several model systems were constructed and simulated, different effects were observed and the possible mechanisms were discussed.Item Cholesterol lowering effects of bovine serum immunoglobulin in human participants with mild hypercholesterolemia(Texas A&M University, 2006-10-30) Black, Melinda LoriHypercholesterolemia is a major risk factor for cardiovascular disease (CVD). Interestingly, the consumption of dairy products, namely milk, has been shown to lower cholesterol. The mechanism of action surrounding this observation has been attributed to the protein fraction of milk. While there have been many studies evaluating the effects of dietary protein sources on cholesterol concentrations, few studies have evaluated specific animal protein components and no human clinical studies regarding the effects of animal plasma protein fractions on cholesterol metabolism have been conducted. This study examined the effect of an oral serum bovine immunoglobulin protein fraction (bIg) derived from US Department of Agriculture approved beef (aged < 30 months) on lipid indices in hypercholesterolemic humans. Participants included men and women (aged 25 ?????? 70 years) with mild hypercholesterolemia (5.44-6.99 mmol/L) who were not receiving cholesterol-lowering medication. Treatment consisted of the randomized, double blind, parallel group, placebo-controlled administration of 5 grams (g) bIg daily for 6 weeks (W) in 52 participants (n = 26 each in treatment and control groups). Mean (???? SD) baseline treatment and placebo total cholesterol (TC) was 6.33 ???? 0.1 mmol/L and 6.16 ???? 0.1 mmol/L respectively. A repeated-measures multivariate analysis of covariance (MANCOVA) covaried for change in total energy and alcohol intake, and a Tukey posthoc examination of the data showed that the bIg-treated group demonstrated a significant reduction in TC at 3-week (W) (5.98 ???? 0.5 mmol/L; P < 0.05) and 6-week (W) (5.97 ???? 0.7 mmol/L; P > 0.05) intervals compared to baseline. The 6W concentration was significantly lower than the placebo group (P < 0.05). Additionally, study findings displayed no significant changes in the placebo group or in any other lipid indexes or markers associated with hepatorenal or cardiovascular health. Consumption of bIg appears to lower major lipid indexes associated with CVD.Item Citrus limonoids and flavonoids: extraction, antioxidant activity and effects on hamster plasma cholesterol distribution(Texas A&M University, 2005-11-01) Yu, JunFour in vitro models were used to measure the antioxidant activity of 11 citrus phytochemicals. The citrus limonoids and bergapten showed very weak antioxidant activity. The flavonoids demonstrated mild, to moderate, to strong antioxidant activity. In addition to some other commonly accepted structural features our data indicated that the hydroxyl group in position 6 of ring A could also increase the antioxidant activity of flavonoids. Compared with the active flavonoids, limonoids are highly oxygenated triterpenoids, with fewer hydroxyl groups to stabilize unpaired electrons (or scavenge free radicals). Bergapten lacks a hydroxyl group. This is the first report on the antioxidant activity of limonoids and neoeriocitrin. A feeding study using Syrian hamsters was followed to determine the effect of citrus limonoids and flavonoids on plasma cholesterol. Hamsters fed with limonin, limonin 17-Beta-D-glucopyranoside and grapefruit pulp significantly inhibited the increase of LDL/HDL-cholesterol (36.6%, 52.9% and 57% respectively) compared with the basal control (65.8%) and the pectin control (70%). Furthermore, hamsters fed with limonin had significantly larger LDL particle size (21.21 nm) compared with the control group (19.96 nm). Further studies demonstrated that LDLs from hamsters fed with limonin and limonin 17-Beta-D-glucopyranoside were less susceptible to oxidation. These data suggest that limonin, limonin 17-Beta-D-glucopyranoside and grapefruit pulp have potential inhibitory effects against atherogenesis. Supercritical CO2 (SC-CO2) was attempted to extract limonoids from grapefruit seeds and molasses. Limonin aglycone was successfully extracted with SC-CO2 directly from grapefruit seeds with the yield of 6.3 mg/g seeds at 48.3 MPa, 50˚C and 60 min with CO2 top feeding; and the limonin glucoside was extracted using SC-CO2 and ethanol as co-solvent from the defatted seeds with the yield of 0.73 mg/g seeds at 42 MPa, 52˚C, 45% ethanol (XEth=0.45) and 40 min with CO2 top feeding; and limonin glucoside also was extracted using SC-CO2 and ethanol with the yield of 0.61mg/g grapefruit molasses at 48.3 MPa, 50˚C and 10% ethanol (XEth=0.1), 40 min with CO2 top feeding. CO2 flow rate was around~5 l/min in experiments. The results demonstrated SC-CO2 extraction of limonoids from citrus juice industry byproducts has practical significance for future commercial production.Item Comparative aspects of cholesterol metabolism and lecithin:cholesterol acyltransferase activity in dogs and cats(2009-05-15) Angell, Rebecca JoyceLittle research has focused on the relationship between lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol metabolism in dogs and cats. To study weight loss and cholesterol metabolism in dogs, four experimental weight-loss diets were fed to 12 obese female beagles for 8 wk in a partial crossover design (n = 6). High- (HGI) or low-glycemic index (LGI) starch and diacylglycerol or triacylglycerol oil were combined to compose diets with similar fatty acid (FA) profiles. Body weight was measured weekly. Fasted blood samples were drawn at wk1, wk4, and wk8 to measure plasma total (TC), unesterified (UC), and esterified cholesterol (EC) concentrations, LCAT activity, and FA composition of the phospholipid (PL) and EC fractions. All groups lost weight. UC increased from wk1 to wk4 (p < 0.05). LCAT activity increased from wk1 to wk4 and remained elevated at wk8 (p < 0.05). Plasma PL FA profiles reflected the diets fed with few diet or time effects. Plasma EC FA profiles reflected the specificity of LCAT for linoleic acid (LA) with minimal diet or time effects. We conclude that weight reduction in dogs occurs in conjunction with increased LCAT activity and altered plasma cholesterol fractions but not changes in plasma PL or EC FA profiles. To measure the activity and demonstrate the FA specificity of LCAT in felines fed varying types of fat, 29 female cats were fed diets enriched with high-oleic sunflower (n = 9), menhaden fish (n = 10), or safflower (n = 10) oil (8g oil/100g kibble) for 4 wk. Fasted blood samples were drawn at d0, d14, and d28 for determination of the blood parameters mentioned previously. LCAT and TC showed no time or diet effects. UC decreased at d28 compared to d0 and d14, while EC increased at d28 compared to d0 and d14 (all p < 0.05). Plasma EC FA profiles reflected the specificity of LCAT for LA with many diet and time effects but contained no docosahexanoic acid (DHA). We conclude that feline LCAT has no measurable affinity for DHA, but both feline and canine LCAT demonstrated specificity for LA regardless of diet fed.Item Development, validation, and application of cholesterol determination method for meat and poultry products using gas chromatography(2010-12) Dinh, Thu T. N.; Thompson, Leslie D.; Brooks, Chance; Galyean, Michael L.; Boylan, Lee M.; Patterson, KristineMichael Brown and Joseph Goldstein, two Nobel laureates, declared cholesterol to be the most highly decorated small molecule in biology, with 13 Nobel Prizes awarded to scientists dedicating their professional careers to cholesterol research. Windaus first determined cholesterol with using digitonin in 1910, followed by Grigaut with a colorimetric method using Liebermann-Burchard reaction in 1911. The development of innovative technologies and analytical instruments has allowed for incredible specificity, sensitivity, accuracy, and precision of cholesterol determination. The AOAC Official Method 994.10 employing GC-FID is currently used in most commercial laboratories for routine analysis of cholesterol in foods. Although significantly modified by employing direct saponification, it is still costly, time-consuming, and labor-intensive. The primary objectives of the present study were to refine the analytical conditions for improved performance, develop a written procedure, and determine intra- and inter-laboratory performances of a newly developed method using the AOAC-required performance measurements. Cholesterol was liberated from meat and poultry samples and was detected using GC-FID at a retention time of 11.96 min. Using 0.005 mg of 5α-cholestane/mL as internal standard, the linearity of the standard curve was determined for peak ratio and standard concentrations of 0.008 to 0.020 mg of cholesterol/mL with a coefficient of determination of 0.995 and a response factor of 0.66 (CV = 3.84%). The limits of detection and quantitation were 1.24 and 4.00 mg/100 g, respectively. Cholesterol was stable in the toluene extract when stored for up to 6 d under refrigeration before GC analysis as demonstrated by high precision (0.92 to 2.69% CV) and accuracy (93.24 to 100.56% recovery) of the results. This method was proved to be more accurate and precise compared with results from two commercial laboratories using the AOAC 994.10 method. The new method was also tested for robustness in two ruggedness trials and was collaboratively validated. This method has now been routinely applied to update cholesterol content of meat and poultry in the USDA National Nutrient Database for Standard Reference with high reliability and productivity. Data from 363 meat and poultry samples with 799 replicates showed that 91.5% of total samples were measured with CV of less than 6% and Horwitz ratios of less than 2.0. Collective data for a meat homogenate, SRM1546 (certified reference material), indicated that most of measured concentrations were within the certified range.Item Direct measurement of membrane dipole field in complex model membranes via vibrational stark effect spectroscopy coupled with molecular dynamics simulations(2016-08) Shrestha, Rebika; Webb, Lauren J.; Elber, Ron; Gordon, Vernita; Stachowiak, Jeanne; Vanden Bout, DavidThe heterogeneous composition of a biological membrane creates a complex electrostatic environment that regulates membrane structure and function. In this work, we investigated the magnitude of the membrane dipole field, Fd, located entirely within the low dielectric membrane interior as a function of membrane composition complexity. We directly measured Fd in vesicle model membrane composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using vibrational Stark effect (VSE) shifts of nitrile oscillators systematically placed along the membrane interior coupled with extensive Molecular Dynamics (MD) simulations. We calculated the absolute magnitude of Fd in DMPC vesicles to be 8-11 MV/cm, at the high end of the range provided in literature. We increased the complexity of the membrane composition by intercalating cholesterol molecule at a wide range of concentration (0- 40 mol%) and found that cholesterol increased Fd at low concentration (~10 mol%), and decreased Fd at higher concentration (>10 mol%). This result, when compared to lipid bilayers containing a cholesterol derivative, 6-ketocholestanol (6-kc) that differs from cholesterol by only a ketone functional group, was strikingly different. Using the spectral line widths obtained from Fourier-transform infrared spectroscopy experiments and molecular dynamic simulations on model lipid-sterol bilayers, we propose that the membrane dipole field is greatly correlated to the local membrane structure and organization regulated by the sterols in the bilayer. We propose that at low concentrations, cholesterol increases dipole field by increasing packing density of disordered lipids, cholesterol and their associated hydrogen bonded water dipoles whereas at high concentrations, the sterol decreases the field by forming liquid ordered state enriched in cholesterol, thus spacing out phospholipids along with water dipoles. 6-kc, on the other hand, is homogeneously distributed and increases hydrogen bonding with water dipoles via two polar groups on its sterol ring, thus never promoting ordered domain and increasing the dipole field monotonously. We also investigated the translocation mechanism of positively, negatively and zwitterionic charged tryptophan molecules through a phospholipid bilayer using time dependent fluorescence spectroscopy and atomically detailed simulations. Both experiment and simulation reproduced the qualitative trend and suggested that the fastest permeation occurred for positively charged tryptophan. Molecular dynamics simulations revealed that the translocation mechanism was assisted by a local defect and the permeation process was insignificantly influenced by the long-range electrostatic interactions, such as the membrane dipole potential.Item Effect of hydroxytyrosol supplementation on the lipid profile and metabolic disease risk markers in healthy men(2012-05) Burns, James Dorsey; Ivy, John, 1945-Hydroxytyrosol (HT) has been found to be a potent antioxidant and hypocholesterolemic agent in various animal models of disease including dyslipidemia, atherosclerosis, and diabetes. Therefore, the purpose of this study was to examine the effects of hydroxytyrosol (HT) supplementation on the lipid profile and metabolic risk markers in recreationally active men. Sixty-one (n = 61) subjects (21.46 ± 0.22 yrs, 179.46 ± 0.79 cm, 78.91 ± 1.19 kg) consumed HT in either a high dose (HI, 150 mg HT; n = 22), a low dose (LO, 50 mg HT; n = 20), or a placebo (PLA; n = 19) every day for 6 weeks. Blood draws were obtained at baseline, 14, 28, and 39 days under fasting conditions. Analyzed were the components of the plasma lipid profile: total cholesterol (TC), high density lipoprotein cholesterol (HDLc), the TC:HDLc fraction, low density lipoprotein cholesterol (LDLc), very low density lipoprotein cholesterol (VLDLc), and triglycerides (Tg); and markers of metabolic risk: uric acid, lipase, hemoglobin (Hb), glycated hemoglobin (HbA1c), and blood glucose (BG). The primary finding was that HT, in either HI or LO dosages did not cause clinically meaningful changes in the blood lipid profile or markers of metabolic risk. Subjects in the HI group experienced a small big significant increase in fasting blood glucose, while those in the PLA group experienced a significant increase in VLDLc concentration. In both cases, however, the mean values remained within their respective healthy reference ranges. Whether these changes would persist beyond the 6-week course of this study is not known. While no improvements were seen in any of our selected measures, these results indicate that HT supplementation, ranging from 50 to 150 mg/day, is safe to consume for durations up to 6 weeks in healthy young men. By maintaining the lipid profile and metabolic risk markers within a healthy range, it is possible that HT may impart a degree of protection against cardiovascular and metabolic disease risk, but such an effect may only be apparent when the plasma lipid and/or metabolic risk profile is abnormal.Item Evaluation of a pharmacist-led medication management program in high-risk diabetic patients: impact on clinical outcomes, medication adherence, and pharmacy costs(2009-12) Hanson, Kristin Anne; Wilson, James P.; Rascati, Karen L.; Godley, Paul J.; Browne, Barry A.Diabetes mellitus is a group of metabolic disorders caused by a relative or absolute lack of insulin. Currently, 23.6 million Americans have diabetes. Diabetes can lead to serious microvascular and macrovascular complications, such as cardiovascular disease, blindness, kidney disease, lower-limb amputations, and premature death. Due to the potential cardiovascular complications and the high prevalence of co-morbid hypertension and/or hyperlipidemia in patients with diabetes, diabetes management should include close monitoring of blood glucose, blood pressure, and cholesterol levels. Medical management of diabetic patients is costly; approximately 1 in every 10 health care dollars is currently spent treating diabetes. Studies have shown that in chronic conditions such as diabetes, increased medication use results in demonstrable improvements in health outcomes, reduced hospitalization rates, and decreased direct health care costs. To date no studies have evaluated the impact of a pharmacist-led intervention on diabetic medication adherence. The purpose of this investigation was to analyze the impact of a pharmacist-led medication management program on medication adherence and pharmacy costs and to evaluate clinical measures of diabetes, hypertension, and hyperlipidemia. This study was a quasi-experimental, longitudinal, pre-post study, with a control group. Scott & White Health Plan (SWHP) patients with diabetes (type 1 or type 2), poor glycemic control (most recent A1C >7.5%), and living within 30 miles of participating pharmacies were invited to participate in the intervention which consisted of monthly appointments with a clinical pharmacist and a co-payment waiver for all diabetes medications and testing supplies. A total of 118 patients met study inclusion criteria and were enrolled in the intervention between August 2006 and July 2008. Intervention patients were matched on sex and age to SWHP patients with poor diabetes control living more than 30 miles from a participating pharmacy. To measure the impact of the intervention, medical and pharmacy data were evaluated for one year before and after the study enrollment date. A significant difference was seen in the percentage of patients with type 1 diabetes in the intervention group (14) and the control group (3). The medication management program significantly improved A1C levels in intervention patients relative to controls (-1.1% vs. 0.6%) and was more effective in lowering A1Cs in type 2 diabetics than type 1 patients. Although the generalized linear model did not show that the intervention significantly improved the percentage of patients achieving the ADA goal A1C of <7% compared to controls, the multivariate logistic regression, which controlled for factors such as diabetes type, showed that patients participating in the intervention were 8.7 times more likely to achieve the A1C goal. Persistence with diabetic medications and the number of medications taken significantly increased in the intervention group; however, adherence rates, as measured by medication possession ratio (MPR), did not significantly improve relative to controls. The expenditure on diabetic medications and testing supplies increased substantially more in the intervention group than in the control group. The percentage of patients adherent with antihypertensive medications (MPR ≥80%) increased from 76% to 91% in the intervention group and decreased from 68% to 63% in the control group (P<0.05); no significant difference in blood pressure control was observed. For hyperlipidemia medications, adherence and persistence increased and pharmacy costs decreased in both groups, likely due to the introduction of the first generic HMG-CoA reductase inhibitor into the market during the study period. Future research is needed on the impact of the intervention on medical resource utilization and costs.Item Fat and cholesterol analyses of selected animal tissues.(Texas Tech University, 1975-05) Persky, Janice DarleneNot availableItem High-Oleic Ground Beef, Exercise, and Risk Factors for Cardiovascular Disease in Men and Postmenopausal Women(2012-02-14) Gilmore, Linda AnneSixty-six percent of the ground beef consumed in the U.S. contains 16-30 percent fat by weight, and at the retail level, ground beef fat varies widely with regards to saturated, monounsaturated and trans-fatty acid content. Through two independent studies the effect of fatty acid composition of ground beef on selected cardiovascular disease risk indicators was evaluated. In the first study, 27 free-living normocholesterolemic men completed a three-way crossover dietary intervention. Subjects consumed five, 114-g ground beef patties per week for 5 wk with intervening 4-wk washout periods. Patties contained 24 percent total fat with monounsaturated fatty acid:saturated fatty acid (MUFA:SFA) of either 0.71 (low-MUFA, pasture-fed), 0.83 (mid-MUFA, short-term corn-fed), or 1.10 (high-MUFA, long-term corn-fed). Blood was collected from each subject before and at the end of each diet period. Overall, the ground beef interventions decreased plasma insulin, HDL2, and HDL3 particle diameter and ?-linolenic acid (18:2 (n-3)), and increased plasma arachidonic (20:4(n-6)). The greatest increase in HDL cholesterol from baseline (0.07 mmol/L) was after the high-MUFA ground beef intervention. An increase from baseline in LDL particle diameter (0.5 nm) occurred after the mid- and high-MUFA interventions.We concluded that low-MUFA ground beef from pasture/hay-fed cattle was no more ?heart healthy? than high-MUFA ground beef from corn-fed cattle as judged by common clinical criteria. In the second study, 19 of 29 post menopausal women completed a two-way crossover design. Subjects consumed five, 114-g ground beef patties per week for 6 wk periods separated by a 4 wk washout period. The low-MUFA patties contained 19.4 percent fat with MUFA:SFA of 0.9. The high-MUFA patties contained 22.5 percent fat with a MUFA:SFA ratio of 1.3. In addition to patty consumption, the subjects completed a bout of exercise during the last week of each phase. Blood was taken before, each diet phase (24 hr before exercise) and 24 hr post exercise. Total cholesterol was increased by the high-MUFA patties with the most significant increase seen in HDL cholesterol, mainly HDL2b subfraction. Lipid-rich lipoprotein fractions were increased with the low-MUFA diet, but not by the high-MUFA diet. Very long chain fatty acids were depressed by low MUFA patty consumption. When unadjusted for plasma volume shifts (raw), exercise decreased triglycerides in all three phases. Raw VLDL cholesterol was reduced after exercise during the intervention phases. Raw RLP and IDL cholesterol were reduced after exercise during the high-MUFA intervention. HDL2b was reduced after exercise during the high-MUFA phase. LDL mean size increased and LDL mean density decreased after exercise during the low-MUFA intervention. HDL mean density increased after exercise during both ground beef interventions. The data indicate that high-oleic ground beef can reduce some cardiovascular disease risk factors and can be a part of a healthful diet. Exercise can have a beneficial impact on cardiovascular disease risk factors independent and in conjunction with ground beef consumption.Item Improving marbling through genetics and feed supplements(Texas Tech University, 2005-12) Anderson, Mark J.; Johnson, Jay W.; Blanton, John R.; Kim, Sung W.Growth and quality are two major concerns to producers of meat animals. Lean growth in meat animal will affect the yield of that animal, and the amount of intramuscular fat will affect the quality of that animal. This thesis contains two studies that involved two different methods of producing an animal with improved growth and meat quality. The first study used animal breeding and genetics to produce a swine line with improved growth, marbling and meat quality. The second study used a feed supplement (Ascophyllum nodosum) to manipulate the processes involved in intramuscular fat deposition to produce greater fat deposition without affecting overall animal performance. Study 1 used two genetic swine lines, a low serum cholesterol (LC) swine line and a modern (M) swine line, that were crossed (LC×LC, LC×M, M×LC, and M×M) to produce a new line with improved weight gain, marbling and overall meat quality. Once weaned animals were penned by cross with three animals per pen. Pigs were weighed every 7 d from birth to end of nursery phase, then every 14 d until harvest at 154 d. Comparison of linear regressions of the LC×M line to the M×M line, and the LC×LC line to the M×LC line found that the LC×M line grew faster (P < 0.05) than the M×M line, and the LC×LC line grew slower (P < 0.05) than the M×LC line. Comparison of linear regressions of the M×M and M×LC lines found that the two lines were not different (P > 0.05) and grew at the same rate. No differences (P > 0.05) were seen in marbling between treatment groups, but LC×LC group tended to have less (P = 0.079) initial juiciness than the M×LC group, and less (P = 0.075) sustained juiciness than both the M×LC and LC×M groups. Offspring from an M line dam had heavier weights at d 14 and from 28-56 d than offspring from LC sows (P < 0.05). Interaction of sire and dam was seen from 0-7 d and from 70-154 d (P < 0.05). From this study two lines were formed with improved growth, but neither had improved marbling and meat quality. Study 2 used English cross steers (n = 32) and heifers (n = 32) that were fed a commercial corn based diet and differentially supplemented with 2% Ascophyllum nodosum to maximized intramuscular fat deposition as determined by quality grade. Cattle were blocked by sex and divided into one control and three treatment groups receiving Ascophyllum nodosum. Treatment 1 (trt 1) received Ascophyllum nodosum from d 36-50 of the feeding period, trt 2 received Ascophyllum nodosum for the last 14 d of the feeding period, and trt 3 which received Ascophyllum nodosum for both d 36-50 and the last 14 d of the feeding period. Cattle were weighted and ultrasounded at the commencement of trial and every 28 d following until they reach an average body weight of 544 kg. No effect for Ascophyllum nodosum supplementation was found on measured performance characteristics. All treatments groups supplemented with Ascophyllum nodosum had higher actual marbling scores (P < 0.05) than controls. Trt 1 was found to have a highest marbling score of 572.5 (P < 0.05) with the control group having the lowest marbling score of 473.75. Trt 1 had a higher (P < 0.05) quality grade than the control group (P < 0.05) and Trt 2 and Trt 3 were not different (P > 0.05) from any other treatment group. Control group had 25% Choice, 62.5% Select; trt 1 had 75% Choice, 18.8% Select; trt 2 had 62.5% Choice, 25% Select and trt 3 had 56.3% Choice and 31.2% Select. Overall, treatment groups had a 39.58 % increase in Choice quality grade and a 37.5 % decrease in Select quality grade when compared to the control group. These two studies revealed that it is difficult to positively affect both lean growth and intramuscular fat. However, through the use of genetic selection and feed supplementation, improvements in lean growth and intramuscular fat deposition can be achieved. Study 1 found that the effect of the dam on growth is often underestimated and more care should be taken when making breeding decisions. Currently many producers are using terminal cross sires to increase the growth of their offspring and the dam lines are bred to have large litters and good mothering ability. Data collected from this trial suggests that the dam also plays an important role in growth, even after lactation. Study two found that the use of Ascophyllum nodosum increases marbling score in English cross cattle without effecting performance. Supplementation from d 36-50 showed the greatest improvement in marbling score. While the mechanism of action for Ascophyllum nodosum as it relates intramuscular fat deposition is unknown the use of Ascophyllum nodosum as a feed supplement can help to improve marbling score in English cross cattle.Item Improving marbling through genetics and feed supplements(2005-12) Anderson, Mark J.; Johnson, Jay W.; Blanton, John R.; Kim, Sung W.Growth and quality are two major concerns to producers of meat animals. Lean growth in meat animal will affect the yield of that animal, and the amount of intramuscular fat will affect the quality of that animal. This thesis contains two studies that involved two different methods of producing an animal with improved growth and meat quality. The first study used animal breeding and genetics to produce a swine line with improved growth, marbling and meat quality. The second study used a feed supplement (Ascophyllum nodosum) to manipulate the processes involved in intramuscular fat deposition to produce greater fat deposition without affecting overall animal performance. Study 1 used two genetic swine lines, a low serum cholesterol (LC) swine line and a modern (M) swine line, that were crossed (LC×LC, LC×M, M×LC, and M×M) to produce a new line with improved weight gain, marbling and overall meat quality. Once weaned animals were penned by cross with three animals per pen. Pigs were weighed every 7 d from birth to end of nursery phase, then every 14 d until harvest at 154 d. Comparison of linear regressions of the LC×M line to the M×M line, and the LC×LC line to the M×LC line found that the LC×M line grew faster (P < 0.05) than the M×M line, and the LC×LC line grew slower (P < 0.05) than the M×LC line. Comparison of linear regressions of the M×M and M×LC lines found that the two lines were not different (P > 0.05) and grew at the same rate. No differences (P > 0.05) were seen in marbling between treatment groups, but LC×LC group tended to have less (P = 0.079) initial juiciness than the M×LC group, and less (P = 0.075) sustained juiciness than both the M×LC and LC×M groups. Offspring from an M line dam had heavier weights at d 14 and from 28-56 d than offspring from LC sows (P < 0.05). Interaction of sire and dam was seen from 0-7 d and from 70-154 d (P < 0.05). From this study two lines were formed with improved growth, but neither had improved marbling and meat quality. Study 2 used English cross steers (n = 32) and heifers (n = 32) that were fed a commercial corn based diet and differentially supplemented with 2% Ascophyllum nodosum to maximized intramuscular fat deposition as determined by quality grade. Cattle were blocked by sex and divided into one control and three treatment groups receiving Ascophyllum nodosum. Treatment 1 (trt 1) received Ascophyllum nodosum from d 36-50 of the feeding period, trt 2 received Ascophyllum nodosum for the last 14 d of the feeding period, and trt 3 which received Ascophyllum nodosum for both d 36-50 and the last 14 d of the feeding period. Cattle were weighted and ultrasounded at the commencement of trial and every 28 d following until they reach an average body weight of 544 kg. No effect for Ascophyllum nodosum supplementation was found on measured performance characteristics. All treatments groups supplemented with Ascophyllum nodosum had higher actual marbling scores (P < 0.05) than controls. Trt 1 was found to have a highest marbling score of 572.5 (P < 0.05) with the control group having the lowest marbling score of 473.75. Trt 1 had a higher (P < 0.05) quality grade than the control group (P < 0.05) and Trt 2 and Trt 3 were not different (P > 0.05) from any other treatment group. Control group had 25% Choice, 62.5% Select; trt 1 had 75% Choice, 18.8% Select; trt 2 had 62.5% Choice, 25% Select and trt 3 had 56.3% Choice and 31.2% Select. Overall, treatment groups had a 39.58 % increase in Choice quality grade and a 37.5 % decrease in Select quality grade when compared to the control group. These two studies revealed that it is difficult to positively affect both lean growth and intramuscular fat. However, through the use of genetic selection and feed supplementation, improvements in lean growth and intramuscular fat deposition can be achieved. Study 1 found that the effect of the dam on growth is often underestimated and more care should be taken when making breeding decisions. Currently many producers are using terminal cross sires to increase the growth of their offspring and the dam lines are bred to have large litters and good mothering ability. Data collected from this trial suggests that the dam also plays an important role in growth, even after lactation. Study two found that the use of Ascophyllum nodosum increases marbling score in English cross cattle without effecting performance. Supplementation from d 36-50 showed the greatest improvement in marbling score. While the mechanism of action for Ascophyllum nodosum as it relates intramuscular fat deposition is unknown the use of Ascophyllum nodosum as a feed supplement can help to improve marbling score in English cross cattle.Item Influence of dietary cholesterol, cholic acid, isethionic acid and methionine on cholesterol and bile acid metabolism in the rat(Texas Tech University, 1977-05) Medina, Evangeline VariasNot availableItem Insig-Mediated Regulation of Mammalian HMG COA Reductase Ubiquitnation and Degradation(2004-12-15) Sever, Navdar; Brown, Michael S.3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGR) catalyzes the conversion of HMG CoA to mevalonate, which is the rate limiting step in the production of cholesterol and numerous nonsterol isoprenoid products. Mammalian HMGR is regulated by transcriptional and post-transcriptional feedback mechanisms. The transcriptional regulation is mediated by sterol regulatory element binding proteins (SREBPs), which are synthesized as inactive precursors in the endoplasmic reticulum (ER) membrane. In the absence of sterols, SREBP cleavage activating protein (SCAP) escorts SREBPs from ER to the Golgi apparatus, where SREBPs are cleaved by site 1 and site 2 proteases so as to release their amino terminal transcription factor domains to the nucleus. Sterols inhibit the exit of SCAP-SREBP complex from the ER by promoting the binding of two related polytopic ER membrane proteins, Insig-1 and Insig-2, to the membrane domain of SCAP. Insig-1, but not Insig-2, is an SREBP target gene, causing Insig-1 levels to drop in the presence of sterols, when it is expected to exert its action. The degradation of HMGR requires both sterols and a nonsterol product of the mevalonate pathway and the eight membrane spanning segments in its amino terminus. The membrane domains of HMGR and SCAP bear sequence similarity prompting the investigation of whether Insig proteins can also bind to HMGR. Indeed, Insig-1 and Insig-2 were found to interact with HMGR in a regulated manner and mediate its proteasomal degradation. This effect can be specifically inhibited by overexpressing the membrane domain of SCAP. Insigs were shown to promote the ubiquitination of HMGR on lysine 248 in the cytoplasmic loop between transmembrane segments 6 and 7. In an attempt to achieve a better understanding of the mechanism by which HMGR is degraded, a genetic approach was developed to select mutant somatic cells that cannot degrade HMGR in the presence of sterols. The isolation and characterization of Chinese hamster ovary cells deficient in Insig-1 confirmed the endogenous requirement of Insig-1 for HMGR degradation and revealed the role of differential regulation of Insig-1 and Insig-2 in terms of SREBP processing. These studies revealed a complex feedback regulatory system governing cholesterol homeostasis.Item Interaction of simvastatin and aerobic exercise on expression of mitochondrial and cardioprotective proteins in skeletal and cardiac muscle tissue(2006-05) Meaney, Mary Patricia; Starnes, Joseph W.Simvastatin is a cholesterol-lowering drug designed to lower cholesterol by inhibiting HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins also inhibit the production of coenzyme Q (CoQ), which shares the same biosynthetic pathway. CoQ is an essential part of the mitochondrial electron transport chain (ETC) and has antioxidant properties. In addition, statins have been shown to effect the expression of antioxidant enzymes and heat shock proteins. Aerobic exercise has also been shown to have an effect on the aforementioned proteins. Statins and aerobicexercise are often co-prescribed by physicians even though the interaction of statins and exercise in heart and skeletal muscle has not been adequately explored. Purpose: To determine the interaction of simvastatin and exercise on CoQ, catalase (CAT), glutathione peroxidase (GPx), Manganese superoxide dismutase (Mn SOD), and heat shock protein 70 (HSP70) in cardiac muscle tissue and the expression of CoQ in the plantaris. Methods: Female 4-mo-old Sprague-Dawley rats were randomly assigned to four treatment groups (N = 15-18/group): sedentary (SED), sedentary treated with simvastatin (SED+SIM), exercise trained (EX), and exercise trained treated with simvastatin (EX+SIM). Rats assigned to simvastatin treated groups received 10 mg simvastatin (Zocor®)/kg body/eight/day for four weeks. Rats assigned to exercise groups were exercised on a treadmill five days/week for four weeks at about 70% VO2max for a duration that was gradually increased to 60 minutes/day. Twenty-four hours after the last session, the animals were euthanized and the heart and both plantaris muscles were removed. Some hearts were perfused for 20 minutes to rinse away blood and others were subjected to an ischemia-reperfusion (I-R) protocol. Left ventricles of IR hearts and the left plantaris were homogenized in ddH2O and lipids were extracted and analyzed for CoQ by high performance liquid chromatography. CAT, GPx, and Mn SOD activity was measured polarographically and HSP70 expression was determined by western blotting of the supernatant of homogenate from the left ventricular tissue of rinsed hearts. Results: A simvastatin main effect was observed on CoQ expression of cardiac and skeletal muscle, and CAT activity of cardiac muscle tissue. Expression of CoQ was decreased while CAT activity was increased following statin treatment. An exercise main effect was observed on CoQ and HSP70 expression of cardiac muscle tissue. Exercise decreased CoQ expression, but increased HSP70 expression in the heart. An interaction effect was observed on both HSP70 expression and Mn SOD activity of cardiac tissue. With respect to HSP70, treatment with simvastatin slightly attenuated an exercise induced increase in HSP70 expression. With respect to Mn SOD, treatment with simvastatin or exercise decreased activity while a combined treatment restored Mn SOD activity to a level similar to that of animals who received no treatment. Conclusion: Treatment with simvastatin or exercise alone results in alterations in the expression of CoQ and HSP70 and activity of CAT, GPx, and Mn SOD. With co-administration, simvastatin and aerobic exercise interact in such a way that maintains one's antioxidant defenses despite impairment the body's ability to synthesize CoQ.Item Mechanistic Dissection of Insig-1, a Master Regulator of Cholesterol Homeostasis(2006-05-15) Gong, Yi; Brown, Michael S.Insigs are polytopic membrane proteins of the endoplasmic reticulum (ER) that regulate lipid synthesis by controlling the sterol-mediated vesicular transportation of sterol regulatory element binding proteins (SREBPs). SREBPs are ER bound transcription factors that form complexes with Scap. In sterol-depleted cells, Scap escorts SREBPs from the ER to the Golgi apparatus, where SREBPs are proteolytically cleaved to liberate the nuclear fragments that activate genes for cholesterol synthesis and uptake. When sterols overaccumulate in cells, the Scap/SREBP complex is retained in the ER by the anchor proteins called Insigs. In this thesis I describe the formation of a complex between Insig-1 and Scap in a sterol regulated fashion which facilitates the ER retention of Scap. To understand the molecular basis of the interactions between Insig-1 and Scap, I use a site-directed mutagenesis approach to select residues in Insig-1 that are essential for Insig-1/Scap complex formation. This study reveals a functional role for the amino acid Asp-205, which is located at the beginning of the fourth loop of Insig-1. Mutation of this aspartic acid to alanine produces an inactive Insig-1 that no longer binds to Scap, and leads to sterol-resistant processing of SREBPs. Mammalian cells express two Insig proteins differ in their mode of control. Insig-1, but not Insig-2, is an SREBP target gene. Also, Insig-1 protein is degraded more rapidly than Insig-2. Thus, Insig-1 is the focus of the study. I further demonstrate that degradation of Insig-1 is regulated by sterols. When ER cholesterol content is low, Insig-1 is ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Sterol-induced binding of Insig-1 to Scap prevents Insig-1 ubiquitination and degradation. The dynamic change in Insig-1 protein stability, together with its transcriptional control by nuclear SREBPs, creates a new model for the convergent inhibition of SREBP processing and cholesterol supply in animal cells. Taken together, these studies established Insig-1 as the master regulator in the cholesterol homeostasis.Item Modalities of Cholesterol Binding and Modulation of the NPC Proteins and Scap(2011-12-14) Motamed, Massoud; Brown, Michael S.Low density lipoproteins (LDL) and related plasma lipoproteins deliver cholesterol to cells by receptor-mediated endocytosis. The lipoprotein is degraded in late endosomes and lysosomes, allowing cholesterol to be released. Export of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes) requires two lysosomal proteins: Niemann-Pick C2 (NPC2), a soluble protein of 132 amino acids; and NPC1, a membrane protein with 13 putative membrane-spanning helices. Recessive loss-of-function mutations in either NPC2 or NPC1 produce NPC disease, which causes death owing to lipid accumulation in lysosomes of liver, brain, and lung. Consistent with their cholesterol export role, NPC2 and NPC1 both bind to cholesterol. The cholesterol binding site on NPC1 is located in the NH2-terminal domain (NTD), which projects into the lysosomal lumen. This domain, designated NPC1 (NTD), can be expressed in vitro as a soluble protein of 240 amino acids that maintains cholesterol binding activity. This thesis studies NPC2 in detail as summarized below. Despite a shared role as cholesterol binding proteins, NPC2 and NPC1 (NTD) bind to cholesterol in opposite orientations. The crystal structures of NPC2 and NPC1 (NTD) have been solved, and NPC2 binds cholesterol with the iso-octyl chain facing the interior of the protein, whereas, NPC1(NTD) binds cholesterol with the 3ß-hydroxyl facing the interior of the protein. Another striking difference is the kinetics of this cholesterol binding. NPC2 binds and releases cholesterol rapidly (half-time < 2 min at 4oC), while NPC1 (NTD) binds cholesterol very slowly (half-time > 2 hr at 4oC). However, NPC2 can stimulate the rate of cholesterol binding to NPC1 (NTD) (>15-fold in vitro). This stimulation of cholesterol binding to NPC1 (NTD) by NPC2 is believed to occur through a direct transfer of cholesterol from NPC2 to NPC1(NTD). Amino acid residues important for binding or transfer of cholesterol on NPC2 were identified through alanine scan mutagenesis. Residues that decreased binding thermodynamics and/or kinetics mapped to areas surrounding the binding pockets on the crystal structures; residues that decreased transfer, but not binding, mapped to discrete surface patches near the exposed residues of the binding pockets. These surface patches may be sites where the two proteins interact to transfer cholesterol. The most deleterious binding mutant was P120S, a residue in the cholesterol binding pocket; the most deleterious transfer mutant was V81D, a residue on the hydrophobic patch extending outward from the cholesterol binding pocket. The above mutants of NPC2 were unable to rescue LDL-stimulated cholesteryl ester synthesis in NPC2-deficient cells, in contrast to wild-type NPC2. Once LDL-derived cholesterol leaves the lysosomes, it is transported to the endoplasmic reticulum (ER), where it serves a regulatory role in cholesterol homeostasis. In the ER, these regulatory functions include activation of acetyl-coenzyme A acetyltransferase (ACAT), allowing for esterification of cholesterol for storage, and regulation of sterol regulatory element–binding protein (SREBP) localization, a transcription factor that regulates key enzymes for cholesterol synthesis. SREBP cleavage-activating protein (Scap) is the switch that controls SREBP, and therefore cholesterol synthesis. Scap senses cholesterol abundance in the ER and acts as an escort protein. In sterol depleted cells, Scap escorts SREBP to the Golgi complex, where two proteases cleave SREBP, thereby releasing its transcriptionally active domain so that it can go to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake. When cholesterol in abundant, the sterol binds to Scap and triggers a conformational change in the protein that prevents it from escorting SREBPs to the Golgi for proteolytic cleavage. Scap is a 1276 amino acid protein that consists of two domains: an N-terminal domain with 8 transmembrane spanning regions and a C-terminal domain that projects into the cytosol and associates with SREBPs. Previous studies have localized the cholesterol-binding activity of Scap to its membrane domain. Studies described in this thesis identify the cholesterol binding pocket in Scap and identify key residues that play an important role in the protein’s responsiveness to cholesterol binding. The first loop region of Scap (hereafter referred to as Scap(Loop1)) was purified as a recombinant protein and found to have cholesterol binding activity. The specificity of this sterol binding was determined through competition studies and shown to be physiologically relevant. Additionally, this binding affinity and specificity was similar to that of the membrane domain of Scap. Subsequently, alanine scan mutagenesis was performed on Scap(Loop1). Through this approach, several mutations of Scap were identified that constitutively adopt the cholesterol-bound state. This data demonstrates that Scap(Loop1) binds to cholesterol and that the binding then helps induce the conformational change required for Scap to anchor SREBP in ER membranes.Item Purification of Native and Recombinant NPC1(2008-12-23) Dale, Jarrod Donald; Goldstein, JosephThe Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoproteinderived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. We encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. Described here is the initial purification of rabbit NPC1 using a classical biochemical approach and an analysis of the sterol binding properties of native and recombinant NPC1. Our purification yielded a membrane-bound 25-HC-binding protein which was purified more than 14,000-fold from rabbit liver membranes. This protein was identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Initial characterization of the sterol binding properties showed specific binding for 25-HC; however, we were unable to demonstrate significant binding of NPC1 to cholesterol using our current experimental conditions. The availability of assays to measure NPC1 sterol binding in vitro may further the understanding of intracellular sterol transport.Item The Regulation of Cholesterol Absorption: Nuclear Hormone Receptors and Niemann-Pick C1 Like 1(2007-12-15) Valasek, Mark Andrew; Tansey, Lourdes MaluCholesterol plays fundamental roles in cellular physiology, but it is also involved in many pathophysiological processes including atherosclerosis, cholelithiasis, and some forms of neurodegenerative disease. The ability of mammals to selectively absorb cholesterol from the diet while largely excluding plant sterols has been known for more than 75 years, but the precise repertoire of molecular events necessary for this process are just beginning to be elucidated. Recently, several candidates have been put forth as putative intestinal cholesterol "permeases" responsible for cholesterol transport across the intestinal brush border. In addition, members of the nuclear receptor superfamily of ligand-activated transcription factors are known to modulate expression of genes involved in cholesterol and bile acid homeostasis, and cholesterol absorption. Therefore, we wanted not only to clarify the essential molecular mechanisms by which cholesterol was absorbed, but also to investigate the potential role of nuclear receptors in regulating essential steps in this process. Here, we show that a candidate component of the cholesterol transport machinery, caveolin-1 (CAV1), is neither required for intestinal cholesterol transport or sensitivity to the novel cholesterol absorption blocking agent, ezetimibe. This rules out a critical role for caveolin-1 and lends further support to the contention that Niemann-Pick C1 like 1 (NPC1L1) is the bona fide intestinal cholesterol permease. Therefore to better understand new ways in which nuclear receptors could regulate cholesterol absorption, we studied nuclear receptor regulation of NPC1L1 and determined that several nuclear receptors could modulate its expression in small intestine, including peroxisome proliferator-activated receptor alpha (PPAR alpha ) and retinoid X receptor (RXR). Thus, cholesterol absorption can be regulated by nuclear receptor modulation of NPC1L1 expression.Item Role of Cholesterol 24-Hydroxylase in Hippocampal Long-Term Potentiation(2009-06-18) Ramirez, Denise Marie O'Donnell; Russell, David W.The mammalian brain contains a disproportionately large percentage of the body's cholesterol, steady-state levels of which are maintained within a narrow range to preserve membrane function. The brain is denied access to circulating lipoproteins by the blood-brain barrier and therefore relies on de novo cholesterol synthesis through the mevalonate pathway to meet the tissue's requirement for this essential lipid. A small amount of brain cholesterol is turned over daily in select neurons by cholesterol 24-hydroxylase, which catalyzes the production of the membrane-permeable oxysterol 24(S)-hydroxycholesterol and represents the major pathway of cholesterol catabolism in this organ. Mice lacking 24-hydroxylase have a decreased rate of brain cholesterol synthesis and exhibit deficiencies in spatial, associative, and motor learning. Hippocampal slices prepared from these mice are unable to support the induction of long-term potentiation, a type of synaptic strengthening thought to underlie learning and memory. The ability of 24-hydroxylase knockout slices to exhibit long-term potentiation can be restored by treatment with geranylgeraniol, an isoprenoid end-product of the mevalonate pathway. Mechanistic insight into the role of geranylgeraniol in long-term potentiation has been revealed by calcium imaging studies in neurons cultured from wild-type and 24-hydroxylase knockout embryos. Neurons from mice lacking 24-hydroxylase have specific defects in N-methyl-D-aspartate (NMDA) receptor function, a subtype of ionotropic glutamate receptor essential for long-term potentiation. The subunit composition of NMDA receptors located in various functional pools is normal in 24-hydroxylase knockout hippocampus, suggesting that geranylgeraniol does not affect expression of NMDA receptors. Localization studies of 24-hydroxylase show the enzyme is predominantly expressed in the endoplasmic reticulum throughout the soma and dendrites of selected hippocampal, cerebellar, and cortical neurons, consistent with a postsynaptic need for cholesterol turnover in neurons of brain regions important for learning and memory. These findings reveal that cholesterol turnover is important to produce a constant supply of geranylgeraniol, which in turn is necessary for the induction of long-term potentiation and presumably learning in mice.