Taiwan cobra (Naja naja atra) cardiotoxins, are composed of 60 amino acids and structurally stabilized by four disulfide bonds to form three loops structure. Cardiotoxins induced hemolysis of red blood cells and form a channel-like structure in inducing phospholipid vesicles leakage. The aim of the present study is to explore the events affect the membrane-damaging activity of cardiotoxin 3 (CTX3). Depletion of cholesterol in membrane of red blood cells using methyl-£]-cyclodextrin (M£]CD) enhanced the hemolytic activity of CTX3. Expectedly, CTX3 showed a low binding capability and lower membrane-damaging activity toward phospholipid vesicles containing cholesterol. Guanidinated of lysine residues and selective trinitrobenzoylation (TNP) at N-terminus of guanidinated CTX3 (Gu-CTX3) insignificantly altered binding capability and membrane-damaging activity of CTX3 regardless of phospholipid vesicles containing cholesterol. The binding of CTX3, Gu-CTX3 and TNP-Gu-CTX3 with phospholipid vesicles was reduced by increasing NaCl concentration. Consistent with these observations, membrane-damaging activity of CTX3, Gu-CTX3 and TNP-Gu-CTX3 were also decreased with increasing NaCl concentration. Unlike that of Gu-CTX3 and TNP-Gu-CTX3, membrane-damaging activity and membrane-induced oligomerization of CTX3 was completely abolished by 1 M NaCl. Fourier transform infrared spectroscopy indicated that CTX3, Gu-CTX3 and TNP-Gu-CTX3 adopted different conformation on binding with phospholipid vesicles and phospholipid/cholesterol vesicles. Taken together, our data indicate that membrane-bound conformation of CTX3 is affected by cholesterol and guanidination of lysine residues.

No description available.

Lysine is of prime nutritional significance since it is the first limiting amino acid in many foods of plant origin and is easily rendered unavailable upon heat processing or upon unfavourable storage conditions. The term "available lysine" refers to forms of lysine which contain free €-amino groups within the peptide chain. Once the Є-amino group is blocked, lysine becomes unavailable since it cannot be hydrolyzed by proteolytic enzymes. The availability of lysines in casein, lysozyme, β-lactoglobulin, acid solubilized gluten and whole egg was determined by the pepsin pancreatin digestion test, the dinitrobenzene sulfonic acid (DNBS) and the trinitro-benzene sulfonic acid (TNBS) methods. The results were compared to the fluorodinitrobenzene (FDNB) method. Good agreement was obtained between the FDNB official method and the DNBS technique, with a correlation coefficient of 0.989. When the TNBS method was compared to the FDNB difference technique, a correlation coefficient of 0.988 was found. The pesin pancreatin digestion test indicated the relative amount of lysine released by the enzymes under the conditions specified by the test. A correlation coefficient of 0.995 was found between the FDNB official method and the enzymatic test. The specificity of DNBS for the Є-amino group of lysine was determined using α- and Є-formyl-lysines, L-lysine, L-lysyllysine, L-lysylalanine and ribonuclease-S-peptide. DNBS was found to react mainly with Є-amino group but with a slight reactivity with α-amino group. However, in the case of proteins with several Є-amino groups and N-terminal lysine, the contribution of the Є-amino group to the results becomes negligible. The DNBS method was found to be the simplest and most reliable method for determination of available lysine, for the following reasons: a) it does not require acid hydrolysis of the proteins; b) a large number of samples can be analyzed simultaneously in a few hours, and c) it does not require expensive and lengthy chromatographic amino acid analysis. / Land and Food Systems, Faculty of / Graduate

Sirtuins, a class of enzymes known as nicotinamide adenine dinucleotide-dependent deacetylases, have been shown to regulate a variety of biological processes, including aging, transcription, and metabolism. Severn human Sirtuins members (Sirt1-7) are involved in various kinds of severe diseases like aging, cancer development, autoimmune diseases and therefore are considered as potential drug targets for treatment. Among them, Sirt4-7 have very weak traditional deacetylation function in contrast to the others. So, investigation on the real functions of these sirtuins is a prerequisite for specific modulator (inhibitor or activator) design. Crystallography is a robust way to study the molecular basis of the catalytic function of these sirtuins. Here we show that the real function of Sirt6 is the de-long-chain-fatty acylase activity from lysine, such as the demyristoylase activity. The crystal structure of Sirt6 complex shows a large hydrophobic pocket accommodating the myristoyl group. Together with the biochemical and physiological data from our collaborators, we confirm that Sirt6 promotes the TNFα secretion via hydrolysis the myristoyl group on K19 and K20. Fatty acylation on lysine occurs in mammalian cells and had been found for years, however, the regulatory mechanism is still unclear. Our results provide the opportunities to understand the regulatory of the long chain fatty acyl modification on lysine via Sirt6, which has been little studied until now. More work will be focused on the structural based development of inhibitors to cure the Sirt6 regulated diseases in the near future. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

No description available.

Typescript. / Bibliography: leaves 117-126. / viii, 126 leaves ill

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains vii, 105 p. : ill. Vita. Includes abstract. Includes bibliographical references.

The effect of a maternal dietary lysine deficiency on milk carnitine levels and on plasma and liver carnitine levels in dams, fetuses and neonates was studied. Experimental animals were fed either a low-lysine diet (0.27% lysine), a high-lysine diet (1.07% lysine) ad libitum, or the high-lysine diet pair-fed to the low-lysine group. All diets contained 20% wheat gluten, 20% corn oil and negligible carnitine. Dams fed a diet, either low in lysine or restricted in total food intake, consumed significantly less food during pregnancy and lactation than high-lysine dams. When compared to high-lysine dams the low-lysine dams and their pair-fed controls gained significantly less weight during pregnancy and lost weight during lactation whereas the high-lysine dams gained weight during lactation. Litter size was not affected by either a dietary lysine deficiency or by the small reduction in total food intake during gestation. However, birth weight of offspring in the low-lysine and high-lysine restricted groups was significantly lower than that of the high-lysine controls. On day 15 of lactation the high-lysine pups weighed significantly more than the high-lysine restricted pups, which in turn weighed significantly mere than the low-lysine pups, suggesting a superior lactation performance for those dams fed the high-lysine control diet and the poorest lactation performance for those dams consuming the low-lysine diet. Liver and heart tissue samples were obtained from dams and their offspring on day 21 of pregnancy and day 15 of lactation. When liver weight or heart weight were expressed as a percentage of total body weight for dams or pups, no significant difference between dietary groups was detected. These results indicate that liver and heart weights were proportional to body weight. The low-lysine diet had no significant effect, on day 21 of gestation, on maternal plasma or liver carnitine levels or on fetal liver carnitine levels, whereas fetal plasma carnitine showed a small but significant increase compared to the high-lysine group. On day 15 of lactation plasma and liver carnitine levels were significantly higher in both dams and offspring fed the low-lysine diet, than in their respective controls. This increase in plasma and liver carnitine levels was probably due to a lowered food intake since animals fed the high-lysine diet pair-fed to the low-lysine group showed the same tissue carnitine response as did animals fed the low-lysine diet. Milk carnitine levels on day 2 of lactation were highest in the high-lysine group and lowest in the high-lysine restricted group. On days 8 and 15 of lactation milk carnitine levels were significantly higher in dams fed the low-lysine diet than in those fed the high-lysine or the high-lysine restricted diet. The results of this research indicate that plasma and liver carnitine levels in both dams and offspring and milk carnitine levels in dams, are not limited by the lysine content of the maternal diet under the experimental conditions of this study. / Land and Food Systems, Faculty of / Graduate

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published_or_final_version / Zoology / Doctoral / Doctor of Philosophy