A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate):D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13 [EC] ] was changedto arginine by site-directed mutagenesis. The k_cat of the resultingmutant protein, K146R, was 500 times slower than wild-type insteady-state kinetic assays for both cleavage and condensationof fructose-1,6-bis(phosphate), while the Km for this substratewas unchanged. Analysis of the rate of formation of catalyticintermediates showed K146R was significantly different fromthe wild-type enzyme and other enzymes mutated at this site.Single-turnover experiments using acid precipitation to trapthe Schiff base intermediate on the wild-type enzyme failedto show a build-up of this intermediate on K146R. However, K146Rretained the ability to form the Schiff base intermediate asshown by the significant amounts of Schiff base intermediatetrapped with NaBH₄. In the single-turnover experiments it appearedthat the Schiff base intermediate was converted to productsmore rapidly than it was produced. This suggested a maximalrate of Schiff base formation of 0.022 s^–1, which wasclose to the value of k_cat for this enzyme. This observationis strikingly different from the wild-type enzyme in which Schiffbase formation is >100 times faster than k_cat. For K146Rit appears that steps up to and including Schiff base formationare rate limiting for the catalytic reaction. The carbanionintermediate derived from either substrate or product, and theequilibrium concentrations of covalent enzyme-substrate intermediates,were much lower on K146R than on the wild-type enzyme. The greaterbulk of the guanidino moiety may destabilize the covalent enzyme-substrateintermediates, thereby slowing the rate of Schiff base formationsuch that it becomes rate limiting. The K146R mutant enzymeis significantly more active than other enzymes mutated at thissite, perhaps because it maintains a positively charged groupat an essential position in the active site or perhaps the Argfunctionally substitutes as a general acid/base catalyst inboth Schiff base formation and in subsequent abstraction ofthe C4-hydroxyl proton.     

Folic acid‐conjugated depolymerized quaternized chitosan as potential targeted gene delivery vector

Ligand‐conjugated delivery vectors that target over‐expressed cell surface receptors have a potential impact on gene therapy. In the study reported, high‐molecular‐weight chitosan was depolymerized to medium and low molecular weight and trimethylated to render the polymer soluble over a wider pH range. Folate conjugation was introduced to the quarternized derivative to improve gene transfection efficiency. Complexes of the folic acid‐conjugated trimethylated depolymerized chitosan (FTMC) with plasmid DNA (pDNA) formed core–shell nanostructured particles. Gel electrophoretic band retardation showed efficient condensation of DNA. These derivatives and their complexes with pDNA were tested for toxicity and haemocompatibility and were found to be significantly less toxic and haemocompatible than polyethyleneimine. Transfection efficiency and nuclear uptake properties were tested in the human KB oral epidermoid cell line, which over‐expresses the folate receptor in the presence of 10% serum. Among the four FTMC derivatives investigated, folic acid‐conjugated chitosan having low molecular weight and medium folate conjugation was found to be a potential vector for gene delivery applications with good transfection and nuclear uptake properties, as proved by YOYO labelling of pDNA. Copyright © 2011 Society of Chemical Industry     

High-Fat Diet Alters Serum Fatty Acid Profiles in Obesity Prone Rats: Implications for In Vitro Studies

High‐fat diets (HFD) are commonly used in rodents to induce obesity, increase serum fatty acids and induce lipotoxicity in various organs. Invitro studies commonly utilize individual free fatty acids (FFA) to study lipid exposure in an effort to model what is occurring in vivo; however, these approaches are not physiological as tissues are exposed to multiple fatty acids in vivo. Here we characterize circulating lipids in obesity‐prone rats fed an HFD in both fasted and fed states with the goal of developing physiologically relevant fatty acid mixtures for subsequent in vitro studies. Rats were fed an HFD (60 % kcal fat) or a control diet (10 % kcal fat) for 3 weeks; liver tissue and both portal and systemic blood were collected. Fatty acid profiles and absolute concentrations of triglycerides (TAG) and FFA in the serum and TAG, diacylglycerol (DAG) and phospholipids in the liver were measured. Surprisingly, both systemic and portal serum TAG were ~40 % lower in HFD‐fed compared to controls. Overall, compared to the control diet, HFD feeding consistently induced an increase in the proportion of circulating polyunsaturated fatty acids (PUFA) with a concomitant decline in monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) in both serum TAG and FFA. The elevations of PUFA were mostly attributed to increases in n‐6 PUFA, linoleic acid and arachidonic acid. In conclusion, fatty acid mixtures enriched with linoleic and arachidonic acid in addition to SFA and MUFA should be utilized for in vitro studies attempting to model lipid exposures that occur during in vivo HFD conditions.     

An investigation of the effect of air addition during oil sand conditioning

Bitumen aeration was studied by flooding samples of medium grade oil sand with de‐ionized water under both ambient and high vacuum conditions. The samples were then agitated on a shaker table. Subsequent analysis revealed the presence or absence of an air gap in the sample container was the single most important factor in determining the overall bitumen recovery. Furthermore, samples that were flooded while under high vacuum produced slightly less bitumen compared to samples flooded at atmospheric pressure. This suggests that oil sand that is not under high vacuum when flooded with water contains some amount of indigenous air (see introduction) that would aid in bitumen flotation.     

DXP Synthase‐Catalyzed C?N Bond Formation: Nitroso Substrate Specificity Studies Guide Selective Inhibitor Design

1‐Deoxy‐D ‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the nonmammalian isoprenoid biosynthetic pathway to form DXP from pyruvate and D ‐glyceraldehyde 3‐phosphate (D ‐GAP) in a thiamin diphosphate‐dependent manner. Its unique structure and mechanism distinguish DXP synthase from its homologues and suggest that it should be pursued as an anti‐infective drug target. However, few reports describe any development of selective inhibitors of this enzyme. Here, we reveal that DXP synthase catalyzes C? N bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal a high affinity of DXP synthase for aromatic nitroso substrates compared to the related ThDP‐dependent enzyme pyruvate dehydrogenase (PDH). Results from inhibition and mutagenesis studies indicate that nitroso substrates bind to E. coli DXP synthase in a manner distinct from that of D ‐GAP. Our results suggest that the incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogues will impart selectivity to DXP synthase inhibitors. As a proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP).     

Variations in Tantalum Carbide Microstructures with Changing Carbon Content

A wide variety of microstructures have been obtained by vacuum plasma spraying (VPS) 39Ta:61C atomic percent feedstock powders. During processing, the powder feed was fed through a high energy VPS plasma plume, where altering nozzle angle changed the overall retained carbon concentration in the deposited material. The samples were subsequently sintered and hot isostatic pressed to homogenize and consolidate the microstructure. The microstructures consisted of grains that were either equiaxed or acicular. In the samples with less carbon loss, the equiaxed grains were either the TaC phase or a TaC matrix that encased fine laths of Ta₄C₃. In the sample with the most carbon loss, acicular grains were found containing layered and parallel TaC, Ta₂C, and Ta₄C₃ laths along the major‐axis of the grains. The phases of the compounds have been determined by using complimentary X‐ray diffraction and electron diffraction techniques. Focused ion beam serial sectioning and transmission electron microscopy tilt series tomography were performed to generate three‐dimensional reconstructions of the microstructure morphologies. This article addresses how tantalum carbide microstructures are controlled by the overall concentration and phase fraction content in each of these samples.     

Off-Optimum Cure of Aerospace Epoxy Adhesives

Four epoxy film adhesives used in aircraft manufacture and repair have been examined to establish the effect of deviation from the cure cycle specified by the manufacturer. In addition to the variation of the cure cycle, two surface preparations of the aluminium adherends (chromic acid etch or grit blast followed by silane treatment) were evaluated. Thermal analysis was used to examine the cure envelope of the adhesive, and its extent of cure and glass transition temperature. The adhesive properties were assessed by shear strength (in both single lap joints and in Iosipescu configuration), durability (Boeing wedge test) and chemical resistance to selected aggressive fluids. The sensitivity of the performance of a particular adhesive to offoptimum cure conditions depends on its composition and needs to be determined, not predicted.     

Inkjet printing of polyvinyl alcohol multilayers for additive manufacturing applications

Here we demonstrate that inkjet printing technology is capable of producing polyvinyl alcohol (PVOH) multilayer structures. PVOH water‐based inks were formulated with the addition of additives such as humectant and pigments. The intrinsic properties of the inks, such as surface tension, rheological behavior, pH, wetting, and time stability were investigated. The ink's surface tension was in the range 30–40 mN/m. All formulated inks displayed a pseudoplastic (non‐Newtonian shear thinning and thixotropic) behavior at low‐shear rates and a Newtonian behavior at high‐shear rates; were neutral solutions (pH7) and demonstrated a good time stability. A proprietary 3D inkjet printing system was utilized to print polymer multilayer structures. The morphology, surface profile, and the thickness uniformity of inkjet printed multilayers were evaluated by optical microscopy and FT‐IR microscopy. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43572.