Supercritical Carbon Dioxide Extraction of Lyophilized Aristotelia chilensis (Mol.) Stuntz Berries as Pre-treatment for Enhanced Anthocyanin Recovery

The supercritical carbon dioxide extraction of lyophilized berries of Aristotelia chilensis (Mol.) Stuntz was studied as possible pre-treatment for enhanced anthocyanin recovery. Effect of pressure, temperature, and process time on the extracted oil yields and on the anthocyanins recovery in the pre-treated fractions were considered. The operating parameters were optimized using the central composite design, and extractions were run in the pressure, temperature, and time ranges of 99.6 to 200.4 bar, 36.6 to 53.4 degrees C, and 0.7-2.3 h. The successive multiple regression analysis indicated pressure and time as major influencing parameters on the extraction yield. Those parameters induced no clear changes in the fatty acid composition of almost all the extracted oils, obtaining an aver-age linoleic acid amount between 35-44 % weight in the lipophilic fractions. Standard-ized methanol extractions demonstrated the influence of the different conditions in the SCO2 pre-treatment processes, resulting in extracted anthocyanin increments ranging from 9 to 26 %

The structure of human 15-lipoxygenase-2 with a substrate mimic

Atherosclerosis is associated with chronic inflammation occurring over decades. The enzyme 15-lipoxygenase-2 (15-LOX-2) is highly expressed in large atherosclerotic plaques, and its activity has been linked to the progression of macrophages to the lipid-laden foam cells present in atherosclerotic plaques.We report here the crystal structure of human 15-LOX-2 in complex with an inhibitor that appears to bind as a substrate mimic. 15-LOX-2 contains a long loop, composed of hydrophobic amino acids, which projects from the amino-terminal membrane-binding domain. The loop is flanked by two Ca2+-binding sites that confer Ca2+-dependent membrane binding. A comparison of the human 15-LOX-2 and 5-LOX structures reveals similarities at the active sites, as well striking differences that can be exploited for design of isoform-selective inhibitors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc

Crystal structure of a lipoxygenase in complex with substrate: The arachidonic acid-binding site of 8R-lipoxygenase

Lipoxygenases (LOX) play critical roles in mammalian biology in the generation of potent lipid mediators of the inflammatory response; consequently, they are targets for the development of isoform-specific inhibitors. The regio- and stereo-specificity of the oxygenation of polyunsaturated fatty acids by the enzymes is understood in terms of the chemistry, but structural observation of the enzyme-substrate interactions is lacking. Although several LOX crystal structures are available, heretofore the rapid oxygenation of bound substrate has precluded capture of the enzyme-substrate complex, leaving a gap between chemical and structural insights. In this report, we describe the 2.0 Å resolution structure of 8R-LOX in complex with arachidonic acid obtained under anaerobic conditions. Subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation. Accompanying experimental work supports a model in which both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery

The structure of human 5-lipoxygenase

The synthesis of both proinflammatory leukotrienes and anti-inflammatory lipoxins requires the enzyme 5-lipoxygenase (5-LOX). 5-LOX activity is short-lived, apparently in part because of an intrinsic instability of the enzyme. We identified a 5-LOX-specific destabilizing sequence that is involved in orienting the carboxyl terminus, which binds the catalytic iron. Here, we report the crystal structure at 2.4 angstrom resolution of human 5-LOX stabilized by replacement of this sequence

The three-dimensional structure of the biotin carboxylase-biotin carboxyl carrier protein complex of E. coli acetyl-CoA carboxylase

Acetyl-coenzyme A (acetyl-CoA) carboxylase is a biotin-dependent, multifunctional enzyme that catalyzes the regulated step in fatty acid synthesis. The Escherichia coli enzyme is composed of a homodimeric biotin carboxylase (BC), biotinylated biotin carboxyl carrier protein (BCCP), and an α2β2 heterotetrameric carboxyltransferase. This enzyme complex catalyzes two half-reactions to form malonyl-coenzyme A. BC and BCCP participate in the first half-reaction, whereas carboxyltransferase and BCCP are involved in the second. Three-dimensional structures have been reported for the individual subunits; however, the structural basis for how BCCP reacts with the carboxylase or transferase is unknown. Therefore, we report here the crystal structure of E. coli BCCP complexed with BC to a resolution of 2.49 Å. The protein-protein complex shows a unique quaternary structure and two distinct interfaces for each BCCP monomer. These BCCP binding sites are unique compared to phylogenetically related biotin-dependent carboxylases and therefore provide novel targets for developing antibiotics against bacterial acetyl-CoA carboxylase. © 2013 Elsevier Ltd

Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663

The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca 2+-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A4 from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation. © FASEB

Substituent effects on the mutagenicity of phenyl glycidyl ethers in Salmonella typhimurium

Phenyl glycidyl ether and 6 para-substituted derivatives, the methoxy, tert-butyl, methyl, chloro, bromo and nitro compounds, were tested in the Ames' test for mutagenicity. With the exception of the tert-butyl derivative in TA1535, all 7 compounds were mutagenic in both strains TA100 and TA1535. Electron-donating groups in the para position decreased mutagenicity while electron-withdrawing groups increased this mutagenicity. The mutagenicity of the series of compounds in both strains could be correlated to the Hammett substituent constants for the para-substituent groups. The glycidyl ether results might best be considered in terms of a normal reaction with bionucleophiles as compared to the literature report for the correlation of mutagenicity with abnormal product formation for a styrene oxide series.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24019/1/0000268.pd

The use of electron beams for pasteurization of meats

Electron beam accelerators can be used for electronic pasteurization of meat products by: (1) using the electrons directly impacting the products, or (2) optimizing the conversion of electron energy to x-rays and treating the product with these x-rays. The choice of process depends on the configuration of the product when it is treated. For electron treatment, ten million electron volt (MeV) kinetic energy is the maximum allowed by international agreement. The depth of penetration of electrons with that energy into a product with density of meat is about five centimeters (cm). Two-sided treatment can be done on products up to 10 cm thick with a two-to-one ratio between minimum and maximum dose. Ground beef patties are about 1.25 cm (0.5 inch thick). Beams with 2.5 MeV electron energy could be used to treat these products. Our calculations show that maximum to minimum dose ratios less than 1.2 can be achieved with this energy if the transverse beam energy is small. If the product thickness is greater than 10 cm, x-rays can provide the needed dose uniformity. Uniform doses can be supplied for pallets with dimensions greater than 1.2 m on each side using x-rays from a 5 MeV electron beam. The efficiency of converting the electron beam to x-rays and configurations to achieve dose uniformity are discussed