No description available.

No description available.

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the stepwise transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) to the amino headgroup of PE. Successive methylation results in the formation of the two intermediates, phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N, N-dimethylethanolamine (PDME), and the final product phosphatidylcholine (PC). PE N-methyltransferase is an integral membrane protein localized primarily in the endoplasmic reticulum (microsomal fraction) of liver. PE-, PMME- and PDME-dependent PE N-methyltransferase activities were purified from Triton X-100 solubilized microsomes 429-, 1542- and 832-fold, respectively. The purified enzyme was composed of a single 18.3 kDal protein as determined by SDS-PAGE. Molecular mass analysis of purified PE N-methyltransferase (in Triton X-100 micelles) by gel filtration on Sephacryl S-300 indicated the enzyme existed as a 24.7 kDal monomer. PE N-methyltransferase catalyzed the complete conversion of PE to PC and had a pH optimum of 10 for all three steps. A Triton X-100 mixed micelle assay was developed to assay PE-, PMME- and PDME-dependent activities of both pure and microsomal PE N-methyltransferase. The AT-terminal amino acid sequence of rat liver PE N-methyltransferase and the recently cloned 23.1 kDal S. cerevisiae PEM 2 were found to be 35% homologous. Double reciprocal plots for PE N-methyltransferase at fixed Triton X-100 concentrations and increasing PE, PMME or PDME were highly cooperative. Similar cooperative effects were noted when phospholipid was fixed and Triton X-100 increased. The cooperativity could be partially abolished if a fixed mol% of nonsubstrate phospholipid such as PC was included in the assay. This would indicate that PE N-methyltransferase has specific binding requirements for a site(s) in contact with the micellar substrate. The occupation of this boundary layer by phospholipid is essential for full expression of enzyme activity. Kinetic analysis revealed that PMME and PDME methylation followed an ordered Bi-Bi mechanism. The overall mechanism involves initial binding of PE to a common site and successive methylation steps involving the binding and release of AdoMet and S-adenosyl-L-homocysteine, respectively. Cysteine residue(s) (which are rapidly oxidized in the absence of reduced thiols) are involved in the catalytic mechanism. Reverse-phase HPLC was used to fractionate the phospholipid products of PE N-methyltransferase into individual molecular species. Substrate specificity experiments on PE N-methyltransferase in vitro and in vivo revealed no selectivity for any molecular species of diacyl PE, PMME or PDME. The PE-derived PC, which is rich in 16:0-22:6, is rapidly remodeled to conform to the molecular species compositon of total hepatocyte PC in vivo . The 18.3 kDal PE N-methyltransferase was found to be a substrate for cAMP-dependent protein kinase in vitro. However, only 0.25 mol phosphorus/mol of PE Af-methyltransferase was incorporated, with no observed effect on activity. Studies on PE N-methyltransferase regulation in choline-deficient rat liver indicated that activity changes were due to elevated levels of cellular PE. Immunoblotting of choline-deficient liver microsomes or hepatocyte membranes with a anti-PE N-methyltransferase antibody revealed no alteration in enzyme mass. While more work is needed, initial indications are that hepatic PE N-methyltransferase is a constitutive enzyme regulated primarily by substrate and product levels. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Gary T Rochelle. Vita. Includes bibliographical references.

No description available.

Thesis (doctoral)--Katholieke Universiteit te Nijmegen. / Bibliography: p. 156-174.

N-acyl ethanolamines (NAEs) are a class of lipids recently recognized as signaling molecules which are controlled, in part, by their degradation by fatty acid amide hydrolase (FAAH). On the basis of previous studies indicating increased NAE levels in a tobacco cell suspension-xylanase elicitor exposure system and the availability of FAAH mutants, overexpressor and knockout (OE and KO) genotypes in Arabidopsis thaliana, further roles of NAEs in A. thaliana plant defense was investigated. The commonly occurring urban antimicrobial contaminant triclosan (TCS) has been shown to suppress lipid signaling associated with plant defense responses. Thus, a second objective of this study was to determine if TCS exposure specifically interferes with NAE levels. No changes in steady state NAE profiles in A. thaliana-Pseudomonas syringae pv. syringae and A. thaliana-flagellin (bacterial peptide, flg22) challenge systems were seen despite evidence that defense responses were activated in these systems. There was a significant drop in enoyl-ACP reductase (ENR) enzyme activity, which catalyzes the last step in the fatty acid biosynthesis pathway in plants, on exposure of the seedlings to TCS at 10 ppm for 24 h and decreased reactive oxygen species (ROS) production due to flg22 in long term exposure of 0.1 ppm and short term exposure of 5 ppm. However, these responses were not accompanied by significant changes in steady state NAE profiles.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrogels prepared from photopolymerization have been widely used in many biomedical applications. Ultraviolet (200-400 nm) or visible (400-800 nm) light can interact with light-sensitive compounds called photoinitiators to form radical species that trigger photopolylmerization. Since UV light has potential to cause cell damage, visible light-mediated photopolymerization has attracted much attention. The conventional method to fabricate hydrogels under visible light exposure requires usage of co-initiator triethanolamine (TEA) at high concentration (∼200 mM), which reduces cell viability. Therefore, the first objective of this thesis was to develop a new method to form poly(ethylene glycol)-diacrylate (PEGDA) hydrogel without using TEA. Specifically, thiol-containing molecules (e.g. dithiothreitol or cysteine-containing peptides) were used to replace TEA as both co-initiator and crosslinker. Co-monomer 1-vinyl-2-pyrrolidinone (NVP) was used to accelerate gelation kinetics. The gelation rate could be tuned by changing the concentration of eosinY or NVP. Variation of thiol concentration affected degradation rate of hydrogels. Many bioactive motifs have been immobilized into hydrogels to enhance cell attachment and adhesion in previous studies. In this thesis, pendant peptide RGDS was incorporated via two methods with high incorporation efficiency. The stiffness of hydrogels decreased when incorporating RGDS. The second objective of this thesis was to fabricate hydrogels using poly(ethylene glycol)-tetra-acrylate (PEG4A) macromer instead of PEGDA via the same step-and-chain-growth mixed mode mechanism. Formation of hydrogels using PEGDA in this thesis required high concentration of macromer (∼10 wt.%). Since PEG4A had two more functional acrylate groups than PEGDA, hydrogels could be fabricated using lower concentration of PEG4A (∼4 wt.%). The effects of NVP concentration and thiol content on hydrogel properties were similar to those on PEGDA hydrogels. In addition, the functionality and chemistry of thiol could also affect hydrogel properties.