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Find video protocols related to scientific articles indexed in Pubmed.
Total syntheses of cis-cyclopropane fatty acids: dihydromalvalic acid, dihydrosterculic acid, lactobacillic acid, and 9,10-methylenehexadecanoic acid.
Org. Biomol. Chem.
PUBLISHED: 10-16-2014
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cis-Cyclopropane fatty acids (cis-CFAs) are widespread constituents of the seed oils of subtropical plants, membrane components of bacteria and protozoa, and the fats and phospholipids of animals. We describe a systematic approach to the synthesis of enantiomeric pairs of four cis-CFAs: cis-9,10-methylenehexadecanoic acid, lactobacillic acid, dihydromalvalic acid, and dihydrosterculic acid. The approach commences with Rh2(OAc)4-catalyzed cyclopropenation of 1-octyne and 1-decyne, and hinges on the preparative scale chromatographic resolution of racemic 2-alkylcycloprop-2-ene-1-carboxylic acids using a homochiral Evan's auxiliary. Saturation of the individual diastereomeric N-cycloprop-2-ene-1-carbonylacyloxazolidines, followed by elaboration to alkylcyclopropylmethylsulfones, allowed Julia-Kocienski olefination with various ?-aldehyde-esters. Finally, saponification and diimide reduction afforded the individual cis-CFA enantiomers.
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Role of chloride in the morphological evolution of organo-lead halide perovskite thin films.
ACS Nano
PUBLISHED: 10-09-2014
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A comprehensive morphological study was used to elucidate chloride's role in CH3NH3PbI3-xClx film evolution on a conducting polymer, PEDOT:PSS. Complex ion equilibria and aggregation in solution, as well as the role they play in nucleation, are found to ultimately be responsible for the unique morphological diversity observed in perovskite films grown in the presence of the chloride ion. An intermediate phase that is generated upon deposition and initial annealing templates continued self-assembly in the case of CH3NH3PbI3-xClx. In the absence of chloride, the film growth of CH3NH3PbI3 is directed by substrate interfacial energy. By employing the through-plane TEM analysis, we gain detailed insight into the unique crystallographic textures, grain structures, and elemental distributions across the breadth of films grown from precursor solutions with different chemistries. The lattice coherence seen in morphologies generated under the influence of chloride provides a physical rational for the enhancement in carrier diffusion length and lifetime.
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The carbohydrate-binding promiscuity of Euonymus europaeus lectin is predicted to involve a single binding site.
Glycobiology
PUBLISHED: 09-10-2014
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Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.
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Binary-metal perovskites toward high-performance planar-heterojunction hybrid solar cells.
Adv. Mater. Weinheim
PUBLISHED: 08-14-2014
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A simple, low temperature solution process for Pb/Sn binary-metal perovskite planar-heterojunction solar cells is demonstrated. Sn inclusion substantially influences the band-gap, crystallization kinetics, and thin-film formation leading to a broadened light absorption and enhanced film coverage on ITO/PEDOT:PSS. As a result, the optimized device shows a PCE exceeding 10%, which is the best result for binary-metal perovskite solar cells so far.
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Copper and silver complexes of tris(triazole)amine and tris(benzimidazole)amine ligands: evidence that catalysis of an azide-alkyne cycloaddition ("click") reaction by a silver tris(triazole)amine complex arises from copper impurities.
Inorg Chem
PUBLISHED: 06-20-2014
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The synthesis and characterization of a silver complex of the tripodal triazole ligand, tris(benzyltriazolylmethyl)amine (TBTA, L(1)), that is used as promoter to enhance Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions is reported. X-ray analysis of the silver(I) complex with L(1) reveals a dinuclear cation, [Ag2(L(1))2](2+), that is essentially isostructural to the copper(I) analogue. While the [Ag2(L(1))2](BF4)2 complex provides catalysis for the azide-alkyne cycloaddition process, evidence is presented that this arises from trace copper contamination. The synthesis of silver(I), copper(II), and copper(I) complexes of a second tripodal ligand, tris(2-benzimidazolymethyl)amine (L(2)), which is used to enhance the rate of CuAAC reactions, is also reported. X-ray crystallography of the Cu(I) complex [Cu(I)3(L(2))2(CH3CN)2](BF4)3 offers structural insight into previous mechanistic speculation about the role of this ligand in the CuAAC reaction.
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Galanin-3 receptor antagonism by SNAP 37889 reduces motivation to self-administer alcohol and attenuates cue-induced reinstatement of alcohol-seeking in iP rats.
J. Pharmacol. Sci.
PUBLISHED: 05-30-2014
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The neuropeptide galanin has a role in promoting alcohol consumption and general feeding behavior. The galanin-3 receptor (GALR3) subtype is implicated in modulating the consumption of alcohol and has therefore been identified as a potential target for new pharmacotherapies to treat alcohol use disorders. We have previously shown that the selective GALR3 antagonist SNAP 37889 reduced voluntary alcohol consumption in iP (alcohol-preferring) rats. The present study firstly aimed to investigate the effect of GALR3 antagonism on the motivational properties of alcohol. Secondly, the potential of GALR3 as a therapeutic target in the prevention of relapse was investigated in response to alcohol-conditioned cues. Administration of SNAP 37889 (30 mg/kg, i.p.) significantly reduced the breakpoint for ethanol under a progressive-ratio operant responding schedule of reinforcement. SNAP 37889 also significantly reduced reinstatement of alcohol-seeking in response to re-exposure to conditioned cues that were previously associated with the availability of alcohol. Collectively, results from the current study provide new evidence of GALR3 involvement in cue-induced relapse and provide further evidence that GALR3 antagonism reduces the motivational drive to consume alcohol. These findings validate further research in to the potential use of SNAP 37889 and other GALR3 antagonists to treat alcohol abuse disorders in humans.
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Dissecting conformational contributions to glycosidase catalysis and inhibition.
Curr. Opin. Struct. Biol.
PUBLISHED: 05-15-2014
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Glycoside hydrolases (GHs) are classified into >100 sequence-based families. These enzymes process a wide variety of complex carbohydrates with varying stereochemistry at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the critical points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallography of enzymes bound to ligands that mimic the critical points, along with advanced computational methods and kinetic isotope effects are discussed.
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Integrated molecular, interfacial, and device engineering towards high-performance non-fullerene based organic solar cells.
Adv. Mater. Weinheim
PUBLISHED: 05-03-2014
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High-performance non-fullerene OSCs with PCEs of up to ca. 6.0% are demonstrated based on PBDTT-F-TT polymer and a molecular di-PBI acceptor through comprehensive molecular, interfacial, and device engineering. Impressive PCEs can also be retained in devices with relatively thick BHJ layer and processed through non-halogenated solvents, indicating these high-performance non-fullerene OSCs are promising for large-area printing applications.
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Experimental and theoretical insights into the mechanisms of sulfate and sulfamate ester hydrolysis and the end products of type I sulfatase inactivation by aryl sulfamates.
J. Org. Chem.
PUBLISHED: 02-20-2014
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Type I sulfatases catalyze the hydrolysis of sulfate esters through S-O bond cleavage and possess a catalytically essential formylglycine (FGly) active-site residue that is post-translationally derived from either cysteine or serine. Type I sulfatases are inactivated by aryl sulfamates in a time-dependent, irreversible, and active-site directed manner consistent with covalent modification of the active site. We report a theoretical (SCS-MP2//B3LYP) and experimental study of the uncatalyzed and enzyme-catalyzed hydrolysis of aryl sulfates and sulfamates. In solution, aryl sulfate monoanions undergo hydrolysis by an S(N)2 mechanism whereas aryl sulfamate monoanions follow an S(N)1 pathway with SO2NH as an intermediate; theory traces this difference to the markedly greater stability of SO2NH versus SO3. For Pseudomonas aeruginosa arylsulfatase-catalyzed aryl sulfate hydrolysis, Brønsted analysis (log(V(max)/K(M)) versus leaving group pK(a) value) reveals ?(LG) = -0.86 ± 0.23, consistent with an S(N)2 at sulfur reaction but substantially smaller than that reported for uncatalyzed hydrolysis (?(LG) = -1.81). Common to all proposed mechanisms of sulfatase catalysis is a sulfated FGly intermediate. Theory indicates a ?26 kcal/mol preference for the intermediate to release HSO4(-) by an E2 mechanism, rather than alkaline phosphatase-like S(N)2 substitution by water. An evaluation of the stabilities of various proposed end-products of sulfamate-induced sulfatase inactivation highlights that an imine N-sulfate derived from FGly is the most likely irreversible adduct.
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Development and application of a novel method for high-throughput determination of PCDD/Fs and PCBs in sediments.
Environ. Toxicol. Chem.
PUBLISHED: 01-31-2014
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A selective pressurized liquid extraction technique was developed for the simultaneous extraction of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) from contaminated sediments. The final method incorporated cleanup adsorbents (Florisil, alumina, and silica) into the extraction cell in a 1:1 ratio of matrix to individual adsorbent (w/w). Sulfur, a common interference found in sediments, was successfully removed by placing activated copper in the extraction bottle prior to extraction. No additional postextraction cleanup was required, and sample throughput was reduced to 2.5?h per sample. Target analytes were quantified using high-resolution gas chromatography/electron-capture negative ionization mass spectrometry and verified by high-resolution gas chromatography/high-resolution mass spectrometry. Though mean analyte recoveries (n?=?3) of PCDD/Fs and dl-PCBs were 84?±?5.8% and 70?±?8.4%, respectively, mean surrogate recoveries for all PCDD/Fs using this novel method were greatly improved compared with US Environmental Protection Agency (USEPA) method 1613 (?25-155%) and USEPA method 8290a (40-135%). After development, the method was used to examine surficial sediment samples from the San Jacinto River waste pits, a Superfund site in Houston, Texas, USA. In all samples, PCDD/Fs and dl-PCBs were detected, and the contaminant concentrations ranged over 5 orders of magnitude.
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MCL and Mincle: C-Type Lectin Receptors That Sense Damaged Self and Pathogen-Associated Molecular Patterns.
Front Immunol
PUBLISHED: 01-01-2014
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Macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle) comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage-associated and pathogen-associated molecular patterns. In this review, we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure-activity relationships of activating ligands, particularly trehalose dimycolate and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies.
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Combined Inhibitor Free-Energy Landscape and Structural Analysis Reports on the Mannosidase Conformational Coordinate.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 09-24-2013
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Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including ?-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.
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Cardioprotective 3,4-dihydroxyflavonol attenuation of JNK and p38(MAPK) signalling involves CaMKII inhibition.
Biochem. J.
PUBLISHED: 09-17-2013
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DiOHF (3,4-dihydroxyflavonol) is cardioprotective against I/R (ischaemia/reperfusion) injury. The biological activities of flavonols are associated with kinase modulation to alter cell signalling. We thus investigated the effects of DiOHF on the activation of MAPKs (mitogen-activated protein kinases) that regulate the cardiac stress response. In an ovine model of I/R, JNK (c-Jun N-terminal kinase), p38(MAPK), ERK (extracellular-signal-regulated kinase) and Akt were activated, and NP202, a pro-drug of DiOHF, reduced infarct size and inhibited JNK and p38(MAPK) activation, whereas ERK and Akt phosphorylation were unaltered. Similarly, in cultured myoblasts, DiOHF pre-treatment preserved viability and inhibited activation of JNK and p38(MAPK), but not ERK in response to acute oxidative and chemotoxic stress. Furthermore, DiOHF prevented stress-activation of the direct upstream regulators MKK4/7 (MAPK kinase 4/7) and MKK3/6 respectively. We utilized small-molecule affinity purification and identified CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) as a kinase targeted by DiOHF and demonstrated potent CaMKII inhibition by DiOHF in vitro. Moreover, the specific inhibition of CaMKII with KN-93, but not KN-92, prevented oxidative stress-induced activation of JNK and p38(MAPK). The present study indicates DiOHF inhibition of CaMKII and attenuation of MKK3/6?p38(MAPK) and MKK4/7?JNK signalling as a requirement for the protective effects of DiOHF against stress stimuli and myocardial I/R injury.
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3,4-Bis-difluoromethoxycinnamoylanthranilate (FT061): An orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathy.
Bioorg. Med. Chem. Lett.
PUBLISHED: 08-05-2013
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Cinnamoylanthranilates including tranilast have been identified as promising antifibrotics that can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. Structure-activity relationships aimed at improving potency and metabolic stability have led to the discovery of FT061. This compound, which bears a bis-difluoromethoxy catechol, attenuates TGF-?-stimulated production of collagen in cultured renal mesangial cells (approx 50% at 3?M). When dosed orally at 20mg/kg to male Sprague Dawley rats, FT061 exhibited a high bioavailability (73%), Cmax of 200?M and Tmax of 150min, and a half-life of 5.4h. FT061 reduced albuminuria when orally dosed in rats at 200mgkg/day in a late intervention study of a rat model of progressive diabetic nephropathy.
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Selective Pressurized Liquid Extraction Technique Capable of Analyzing Dioxins, Furans, and PCBs in Clams and Crab Tissue.
Bull Environ Contam Toxicol
PUBLISHED: 07-10-2013
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A selective pressurized liquid extraction technique (SPLE) was developed for the analysis of polychlorodibenzo-p-dioxins, polychlorodibenzofurans (PCDD/Fs) and dioxin-like polychlorobiphenyls (dl-PCBs) in clam and crab tissue. The SPLE incorporated multiple cleanup adsorbents (alumina, florisil, silica gel, celite, and carbopack) within the extraction cell. Tissue extracts were analyzed by high resolution gas chromatography coupled with electron capture negative ionization mass spectrometry. Mean recovery (n = 3) and percent relative standard deviation for PCDD/Fs and dl-PCBs in clam and crabs was 89 ± 2.3 and 85 ± 4.0, respectively. The SPLE method was applied to clams and crabs collected from the San Jacinto River Waste Pits, a Superfund site in Houston, TX. The dl-PCBs concentrations in clams and crabs ranged from 50 to 2,450 and 5 to 800 ng/g ww, respectively. Sample preparation time and solvents were reduced by 92 % and 65 %, respectively, as compared to USEPA method 1613.
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A click chemistry approach to 5,5-disubstituted-3,3-bisisoxazoles from dichloroglyoxime and alkynes: luminescent organometallic iridium and rhenium bisisoxazole complexes.
J. Org. Chem.
PUBLISHED: 07-02-2013
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5,5-Disubstituted-3,3-bisisoxazoles are prepared in one step by the dropwise addition of aqueous potassium hydrogen carbonate to a mixture of dichloroglyoxime and terminal alkynes. The reaction exhibits a striking preference for the 5,5-disubstituted 3,3-bisisoxazole over the 4,5-regioisomer. Organometallic iridium and rhenium bisisoxazole complexes are luminescent with emission wavelengths varying depending upon the identity of the 5,5-substituent (phenyl, butyl).
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Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins.
Cell. Mol. Life Sci.
PUBLISHED: 02-18-2013
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Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following ?-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.
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Halide-ion-templated Ag8Cu6 rhombic dodecahedrons: synthesis, structure and reactivity of [Ag8Cu6(C?CtBu)12X]BF4 (X = Cl, Br).
Dalton Trans
PUBLISHED: 02-05-2013
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Halide-templated tert-butylethynide Ag14 complexes undergo reversible exchange with copper(I) to afford a series of mixed Ag14-nCun (n = 1-6) species. Up to six silver atoms in the cluster can be replaced with copper(I) to give a core structure featuring a Ag8Cu6 rhombic dodecahedron. These clusters are useful models for investigating the chemical reactivity and photoluminescence of heterometallic d(10)-d(10) systems.
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Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.
J. Org. Chem.
PUBLISHED: 01-30-2013
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Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.
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Cancer risk from incidental ingestion exposures to PAHs associated with coal-tar-sealed pavement.
Environ. Sci. Technol.
PUBLISHED: 01-03-2013
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Recent (2009-10) studies documented significantly higher concentrations of polycyclic aromatic hydrocarbons (PAHs) in settled house dust in living spaces and soil adjacent to parking lots sealed with coal-tar-based products. To date, no studies have examined the potential human health effects of PAHs from these products in dust and soil. Here we present the results of an analysis of potential cancer risk associated with incidental ingestion exposures to PAHs in settings near coal-tar-sealed pavement. Exposures to benzo[a]pyrene equivalents were characterized across five scenarios. The central tendency estimate of excess cancer risk resulting from lifetime exposures to soil and dust from nondietary ingestion in these settings exceeded 1 × 10(-4), as determined using deterministic and probabilistic methods. Soil was the primary driver of risk, but according to probabilistic calculations, reasonable maximum exposure to affected house dust in the first 6 years of life was sufficient to generate an estimated excess lifetime cancer risk of 6 × 10(-5). Our results indicate that the presence of coal-tar-based pavement sealants is associated with significant increases in estimated excess lifetime cancer risk for nearby residents. Much of this calculated excess risk arises from exposures to PAHs in early childhood (i.e., 0-6 years of age).
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A practical synthesis of long-chain iso-fatty acids (iso-C12-C19) and related natural products.
Beilstein J Org Chem
PUBLISHED: 01-01-2013
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A gram-scale synthesis of terminally-branched iso-fatty acids (iso-C12-C19) was developed commencing with methyl undec-10-enoate (methyl undecylenate) (for iso-C12-C14) or the C15 and C16 lactones pentadecanolide (for iso-C15-C17) and hexadecanolide (for iso-C18-C19). Central to the approaches outlined is the two-step construction of the terminal isopropyl group through addition of methylmagnesium bromide to the ester/lactones and selective reduction of the resulting tertiary alcohols. Thus, the C12, C17 and C18 iso-fatty acids were obtained in three steps from commercially-available starting materials, and the remaining C13-C16 and C19 iso-fatty acids were prepared by homologation or recursive dehomologations of these fatty acids or through intercepting appropriate intermediates. Highlighting the synthetic potential of the iso-fatty acids and various intermediates prepared herein, we describe the synthesis of the natural products (S)-2,15-dimethylpalmitic acid, (S)-2-hydroxy-15-methylpalmitic acid, and 2-oxo-14-methylpentadecane.
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Copper(I)-catalyzed cycloaddition of silver acetylides and azides: incorporation of volatile acetylenes into the triazole core.
Org. Biomol. Chem.
PUBLISHED: 07-12-2011
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Silver acetylides and organic azides react under copper(I) catalysis to afford 1,4-disubstituted 1,2,3-triazoles. Mechanistic studies implicate a process involving transmetallation to copper acetylides prior to cycloaddition. This work demonstrates that silver acetylides serve as suitable precursors for entry into copper-mediated coupling reactions. This methodology allows the incorporation of volatile and difficult-to-handle acetylenes into the triazole core.
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Antioxidant activity contributes to flavonol cardioprotection during reperfusion of rat hearts.
Free Radic. Biol. Med.
PUBLISHED: 06-07-2011
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The mechanism of flavonol-induced cardioprotection is unclear. We compared the protective actions of a flavonol that inhibits calcium utilization and has antioxidant activity, 3,4-dihydroxyflavonol (DiOHF); a flavonol that affects only calcium activity, 4-OH-3-OCH(3)-flavonol (4-OH-3-OCH(3)F); and a water-soluble flavonol with selective antioxidant activity, DiOHF-6-succinamic acid (DiOHF-6-SA), in isolated, perfused rat hearts. Hearts were subjected to global ischemia for 20 min followed by 30 min reperfusion and were treated with vehicle (0.05% DMSO), DiOHF, 4-OH-3-OCH(3)F, or DiOHF-6-SA (all 10 ?M, n=5-8 per group). Flavonols were infused for 10 min before ischemia and during reperfusion. In vehicle-treated hearts, left-ventricular (LV) +dP/dt was reduced by 60% at the end of reperfusion compared to the preischemic level. Lactate dehydrogenase (LDH) release was elevated and endothelial NO synthase (eNOS) expression was lower in vehicle-treated hearts compared to shams. In comparison, DiOHF treatment improved LV function upon reperfusion, decreased LDH, and preserved eNOS expression. The antioxidant DiOHF-6-SA also preserved contractility, reduced LDH, and preserved eNOS expression. In contrast, hearts treated with 4-OH-3-OCH(3)F showed a degree of contractile impairment similar to that of the vehicle group. DiOHF and DiOHF-6-SA also exerted cardioprotection when given only during reperfusion and not when administered only before ischemia. Flavonol-induced cardioprotection relies on antioxidant activity and is mainly exerted during reperfusion.
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A semi-invariant V?10+ T cell antigen receptor defines a population of natural killer T cells with distinct glycolipid antigen-recognition properties.
Nat. Immunol.
PUBLISHED: 02-24-2011
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Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(?)14-J(?)18) T cell antigen receptor (TCR) ?-chain and recognition of the glycolipid ?-galactosylceramide (?-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of ?-GalCer-reactive NKT cells that expressed a canonical V(?)10-J(?)50 TCR ?-chain, which showed a preference for ?-glucosylceramide (?-GlcCer) and bacterial ?-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCR? sequence identity, the V(?)10 TCR-CD1d-?-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-?-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.
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An initial probabilistic hazard assessment of oil dispersants approved by the United States National Contingency Plan.
Environ. Toxicol. Chem.
PUBLISHED: 02-22-2011
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Dispersants are commonly applied during oil spill mitigation efforts; however, these industrial chemicals may present risks to aquatic organisms individually and when mixed with oil. Fourteen dispersants are listed on the U.S. Environmental Protection Agency (U.S. EPA) National Oil and Hazardous Substances Pollution Contingency Plan (NCP). Availability of environmental effects information for such agents is limited, and individual components of dispersants are largely proprietary. Probabilistic hazard assessment approaches including Chemical Toxicity Distributions (CTDs) may be useful as an initial step toward prioritizing environmental hazards from the use of dispersants. In the present study, we applied the CTD approach to two acute toxicity datasets: NCP (the contingency plan dataset) and DHOS (a subset of NCP listed dispersants reevaluated subsequent to the Deepwater Horizon oil spill). These datasets contained median lethal concentration (LC50) values for dispersants alone and dispersant:oil mixtures, in two standard marine test species, Menidia beryllina and Mysidopsis bahia. These CTDs suggest that dispersants alone are generally less toxic than oil. In contrast, most dispersant:oil mixtures are more toxic than oil alone. For the two datasets (treated separately because of differing methodologies), CTDs would predict 95% of dispersant:oil mixtures to have acute toxicity values above 0.32 and 0.76 mg/L for Mysidopsis and 0.33 mg/L and 1.06 mg/L for Menidia (for DHOS and NCP, respectively). These findings demonstrate the utility of CTDs as a means to evaluate the comparative ecotoxicity of dispersants alone and in mixture with different oil types. The approaches presented here also provide valuable tools for prioritizing prospective and retrospective environmental assessments of oil dispersants.
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Synthesis of a hypoxia-targeted conjugate of the cardioprotective agent 3,4-dihydroxyflavonol and evaluation of its ability to reduce ischaemia/reperfusion injury.
Bioorg. Med. Chem. Lett.
PUBLISHED: 02-11-2011
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3,4-Dihydroxyflavonol (DiOHF) is a cardioprotective flavonol that reduces injury associated with myocardial ischaemia and reperfusion. We hypothesized that the efficacy of DiOHF could be enhanced through its targeting to hypoxic regions of partial reperfusion. Copper(I)-catalyzed ligation of an azide-modified DiOHF analogue to 2-propargyl-nitroimidazole afforded a DiOHF-nitroimidazole conjugate (DiOHF-NIm). When incubated with Con8 cells under normoxic conditions DiOHF-NIm could be detected in both the culture supernatant and cell lysate, whereas under hypoxic conditions it was present in substantially reduced amounts consistent with its selective metabolism under hypoxia. DiOHF-NIm possessed antioxidant activity comparable to DiOHF through scavenging of superoxide produced by NADPH/NADPH oxidase, but had significantly attenuated vasorelaxant activity. DiOHF-NIm treatment significantly reduced lactate dehydrogenase release following ischaemia/reperfusion in hindlimbs of anaesthetized rats (p <0.05), to a level similar to DiOHF treatment but also at earlier time points. DiOHF-NIm significantly reduced levels of myeloperoxidase (p <0.05), a biomarker of neutrophil accumulation, whereas the reduction afforded by DiOHF was not significant. DiOHF-NIm therefore represents a promising potential therapeutic for ischaemia/reperfusion injury.
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Synthesis of glycoconjugate fragments of mycobacterial phosphatidylinositol mannosides and lipomannan.
Beilstein J Org Chem
PUBLISHED: 01-18-2011
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Mycobacterium tuberculosis, the causitive agent of tuberculosis (TB), possesses a complex cell wall containing mannose-rich glycophospholids termed phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). These glycophospholipids play important roles in cell wall function and host-pathogen interactions. Synthetic PIM/LM/LAM substructures are useful biochemical tools to delineate and dissect the fine details of mannose glycophospholipid biosynthesis and their interactions with host cells. We report the efficient synthesis of a series of azidooctyl di- and trimannosides possessing the following glycan structures: ?-Man-1,6-?-Man, ?-Man-1,6-?-Man-1,6-?-Man, ?-Man-1,2-?-Man-1,6-?-Man and 2,6-di-(?-Man)-?-Man. The synthesis includes the use of non-benzyl protecting groups compatible with the azido group and preparation of the branched trisaccharide structure 2,6-di-(?-Man)-?-Man through a double glycosylation of a 3,4-butanediacetal-protected mannoside. The azidooctyl groups of these synthetic mannans were elaborated to fluorescent glycoconjugates and squaric ester derivatives useful for further conjugation studies.
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Application of chemical toxicity distributions to ecotoxicology data requirements under REACH.
Environ. Toxicol. Chem.
PUBLISHED: 01-01-2011
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The European Unions REACH regulation has further highlighted the lack of ecotoxicological data for substances in the marketplace. The mandates under REACH (registration, evaluation, authorization, and restriction of chemicals) to produce data and minimize testing on vertebrates present an impetus for advanced hazard assessment techniques using read-across. Research in our group has recently focused on probabilistic ecotoxicological hazard assessment approaches using chemical toxicity distributions (CTDs). Using available data for chemicals with similar modes of action or within a chemical class may allow for selection of a screening point value (SPV) for development of environmental safety values, based on a probabilistic distribution of toxicity values for a specific endpoint in an ecological receptor. Ecotoxicity data for acetylcholinesterase inhibitors and surfactants in Daphnia magna and Pimephales promelas were gathered from several data sources, including the U.S. Environmental Protection Agencys ECOTOX and Pesticides Ecotoxicity databases, the peer-reviewed literature, and the Human and Environmental Risk Assessment (HERA) project. Chemical toxicity distributions were subsequently developed, and the first and fifth centiles were used as SPVs for the development of screening-predicted no-effect concentrations (sPNECs). The first and fifth centiles of these distributions were divided by an assessment factor of 1,000, as recommended by REACH guidance. Use of screening values created using these techniques could support the processes of data dossier development and environmental exposure assessment, allowing for rigorous prioritization in testing and monitoring to fill data gaps.
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Comprehensive two-dimensional gas chromatography, retention indices and time-of-flight mass spectra of flavonoids and chalcones.
J Chromatogr A
PUBLISHED: 07-27-2010
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The applicability of comprehensive two-dimensional gas chromatography (GC×GC) for flavonoids analysis was investigated by separation and identification of flavonoids in standards, and a complex matrix natural sample. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of flavonoids was compared on two complementary column sets. Whilst the BPX5/BPX50 (NP/P) column set offers better overall separation, BPX50/BPX5 (P/NP) provides better peak shape and sensitivity. Comparison of the identification power of GC×GC-TOFMS against both the NIST05 MS library and a laboratory (created in-house) TOFMS library was carried out on a flavonoid mixture. The basic retention index information on high-performance capillary columns with a non-polar stationary phase was established and database of mass spectra of trimethylsilyl derivatives of flavonoids was compiled. TOFMS coupled to GC×GC enabled satisfactory identification of flavonoids in complex matrix samples at their LOD over a range of 0.5-10 ?g/mL. Detection of all compounds was based on full-scan mass spectra and for each compound a characteristic ion was chosen for further quantification. This study shows that GC×GC-TOFMS yields high specificity for flavonoids derived from real natural samples, dark chocolate, propolis, and chrysanthemum.
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Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B and evidence for their widespread occurrence in Eucalyptus.
Carbohydr. Res.
PUBLISHED: 07-02-2010
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Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K(1) and K(2). Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C to be present in most, of the species examined. The widespread occurrence of these carbohydrate monoterpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to secretory cavity lumena.
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Effects of 3,4-dihydroxyflavonol on vascular contractions of rat aortic rings.
Clin. Exp. Pharmacol. Physiol.
PUBLISHED: 03-30-2010
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1. 3,4-Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti-oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions. 2. Contractile and relaxation responses were determined in rat endothelium-denuded aortic rings mounted in organ baths. In addition, the phosphorylation of myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17) was determined using western blot analysis. Levels of GTP RhoA, as a marker of RhoA activation, were also measured. 3. Cumulative addition of increasing concentrations of NaF (3.0-12.0 mmol/L) or U46619 (1.0-1000 nmol/L) concentration-dependently increased vascular tension. These responses were inhibited by pretreatment of aortic rings with DiOHF (10, 30 or 100 micromol/L), which dose-dependently decreased vascular contractions induced by 8.0 mmol/L NaF, 30 nmol/L U46619, 0.1 micromol/L phenylephrine and 50 mmol/L KCl. 4. The K(+) channel blockers 4-aminopyridine (3 mmol/L), charybdotoxin (10 nmol/L), apamin (500 nmol/L) and glibenclamide (10 micromol/L) had no effect on vascular relaxation induced by DiOHF (1-30 micromol/L). 5. At 30 micromol/L, DiOHF decreased the activation of RhoA and subsequent phosphorylation of MYPT1, CPI-17 and MLC(20) to almost basal levels. 6. In conclusion, DiOHF decreases vascular contraction at least partly by inhibition of the RhoA/Rho-kinase pathway in rat endothelium-denuded aorta. These results suggest that DiOHF may have therapeutic potential in the treatment of cardiovascular diseases.
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The galanin-3 receptor antagonist, SNAP 37889, reduces operant responding for ethanol in alcohol-preferring rats.
Regul. Pept.
PUBLISHED: 02-23-2010
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The galanin-3 receptor (GALR3) subtype has been identified as having a role in both feeding behaviour and the regulation of emotional states including anxiety. Despite the evidence for an association between galanin and alcohol, the current study is the first to explore the direct role of GALR3 in this context. The present study investigated the potential of the novel selective GALR3 antagonist, SNAP 37889, to reduce anxiety-like behaviour and voluntary ethanol consumption in the iP (alcohol-preferring) rat. This was achieved through a number of behavioural paradigms testing for anxiety, along with the operant self-administration model.
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Interpreting REACH guidance in the determination of the derived no effect level (DNEL).
Regul. Toxicol. Pharmacol.
PUBLISHED: 02-17-2010
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Under the new European chemicals regulation, REACH, a new safety value, the Derived No Effect Level (DNEL) must be established for all chemicals manufactured, imported or used in the EU in quantities greater than 10 metric tonnes per year. The DNEL is to be calculated for all relevant exposure pathways, exposure populations, and endpoints of toxicity. The EU has published guidance on how to derive the DNEL, but this guidance has yet to be put into practice and is in some places not prescriptive. Using the Agency for Toxic Substances and Disease Registry (ATSDR) dataset, we have determined inhalation DNELs for styrene. In doing so, we considered what effect key decisions would have on the calculated DNEL. The resulting DNELs were then compared to existing risk criteria values or occupational exposure limits. General population DNELs were generally more conservative than analogous risk criteria (ranging from approximately 0.05 to 2.5 ppm). Worker DNELs are lower than existing occupational standards (ranging from approximately 0.4 to 20 ppm). To our knowledge, this work represents the first rigorous application and interpretation of the EU guidance for determination of a DNEL and will prove useful as a model for determination of other DNELs under REACH.
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2,6-Disubstituted benzoates as neighboring groups for enhanced diastereoselectivity in beta-galactosylation reactions: synthesis of beta-1,3-linked oligogalactosides related to arabinogalactan proteins.
J. Org. Chem.
PUBLISHED: 11-26-2009
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Arabinogalactan proteins (AGPs) are plant glycoproteins which contain a beta-1,3-linked galactan core. The synthesis of the beta-galactopyranose-1,3-beta-galactopyranose linkage using various 2-O-acyl-protected glycosyl donors has been plagued with poor stereoselectivity and side reactions including orthoester formation and transesterification of the 2-O-acyl group from the donor to the acceptor. We have investigated the use of 2,6-disubstituted benzoyl groups as bulky neighboring groups on the glycosyl donor. A 2,4,6-trimethylbenzoyl group was found to be optimal and enabled the formation of the beta-galactopyranose-1,3-beta-galactopyranose linkage to disarmed ester-protected acceptors, suppressing transesterification and reducing orthoester formation while enhancing the beta-selectivity of galactosylation reactions. A series of beta-1,3-linked oligogalactosides were prepared and elaborated to neoglycoconjugates for the study of AGP biosynthesis and AGP binding proteins.
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The European Unions REACH regulation: a review of its history and requirements.
Crit. Rev. Toxicol.
PUBLISHED: 08-05-2009
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In 2006, the European Union (EU) promulgated a monumental regulatory initiative for the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH). To date, several thousand pages of text have been needed to describe the expectations of this regulation. There were numerous reasons for the promulgation of REACH, but, by and large, it is an extension of the global desire to produce fewer industrial chemicals, to understand the possible human and ecological hazards of those that are produced, and to insure that any major threat is anticipated, as well as prevented. Most industry-related groups consider it the most wide-ranging and costly regulatory initiatives related to health risk assessment ever to be promulgated. This review presents a description of REACH that should inform scientists, managers, and others about its objectives and the means to satisfy them. Registration is required for all chemicals manufactured or imported into the EU, unless specifically exempted. Registration is expected to be a collaborative process among companies, which will generate a dossier containing data on physicochemical characteristics, as well as toxicological and ecotoxicological properties. Though the magnitude of the gaps in the data required for registration is uncertain at this point, it is clear that basic toxicology testing will have to be conducted for many chemical substances that have not undergone formal review up to this point. For many chemicals, an examination of hazards and risks arising from the use of these substances will also be required in the form of a chemical safety report (CSR). Beginning with the dual processes of dossier and substance evaluation, the European Chemicals Agency (ECHA), the Member States of the EU, and the European Commission will identify chemicals that may pose unacceptable hazards to human health and/or the environment, and will curtail or restrict their usage. The implementation of REACH will expand and deepen the fields of applied toxicology and exposure assessment by spurring activity and innovation in sampling and analysis, toxicology testing, exposure modeling, alternative toxicity testing, and risk assessment practices.
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Evaluation and optimization of antifibrotic activity of cinnamoyl anthranilates.
Bioorg. Med. Chem. Lett.
PUBLISHED: 07-21-2009
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Tranilast is an anti-inflammatory drug in use for asthma and atopic dermatitis. In studies over the last decade it has been revealed that tranilast can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. We report a structure-activity study aimed at optimizing the antifibrotic activity of tranilast. A series of cinnamoyl anthranilates were prepared and assessed for their ability to prevent TGF-beta-stimulated production of collagen in cultured renal mesangial cells. We reveal derivatives with improved potency and reduced cellular toxicity relative to tranilast. 3-Methoxy-4-propargyloxycinnamoyl anthranilate reduces albuminuria in a rat model of progressive diabetes, and thus has potential as an innovative treatment for diabetic nephropathy.
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Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont.
Nat. Chem. Biol.
PUBLISHED: 06-10-2009
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Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are alpha-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca(2+)-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca(2+) plays a key catalytic role in helping distort the mannoside away from its ground-state (4)C(1) chair conformation toward the transition state.
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Non-volatile components of the essential oil secretory cavities of Eucalyptus leaves: discovery of two glucose monoterpene esters, cuniloside B and froggattiside A.
Phytochemistry
PUBLISHED: 03-20-2009
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The essential oils extracted from the embedded foliar secretory cavities of many Eucalyptus species are of economic value as pharmaceuticals and fragrance additives. Recent studies have indicated that Eucalyptus secretory cavities may not be exclusively involved in the biosynthesis and storage of essential oils. Therefore, we selected three species upon which to perform an examination of the contents of foliar secretory cavities: Eucalyptus froggattii, E. polybractea and E. globulus. This paper describes the isolation and structural characterization of two non-volatile glucose monoterpene esters, which we have named cuniloside B and froggattiside A, from within the secretory cavities of these species, and shows the presence of these compounds in solvent extracts of the leaves from two other species of Eucalyptus. Both compounds were found in high proportions relative to the essential oils extracted from the leaves. We propose that many other carbohydrate monoterpene esters previously isolated from bulk leaf extracts of various Eucalyptus species may also be localized within the non-volatile fraction of foliar secretory cavities.
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Levels of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in southern Mississippi catfish and estimation of potential health risks.
Chemosphere
PUBLISHED: 03-19-2009
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Fish consumption has been classified as one of the primary pathways of exposure to polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs) and biphenyls (PCBs). In this study, we evaluated tissue levels of the 17 laterally substituted PCDD/Fs, 12 dioxin-like PCBs, and 97 non-dioxin-like PCBs in a number of wild-caught and farm-raised catfish collected throughout southern Mississippi. Total lipid-adjusted TEQ and non-dioxin-like PCB concentrations in wild-caught catfish fillets were significantly higher than concentrations in farm-raised fillet samples. The percent contribution of PCDDs, PCDFs, and dioxin-like PCBs to mean total TEQ varied between wild-caught and farm-raised samples as well as by collection site for wild-caught catfish. The non-dioxin-like PCBs that contributed the most to total PCB concentrations also differed between wild-caught and farm-raised samples. Regardless of whether samples were farm-raised or wild-caught, estimated cancer risks associated with consumption of these catfish were less than 27.0E-06. Overall, results of this study indicate that levels of dioxin-like compounds and PCBs in Mississippi catfish are similar to those measured in more recent studies in the US and that levels of these compounds appear to be decreasing in this food source.
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Aryl sulfamates are broad spectrum inactivators of sulfatases: effects on sulfatases from various sources.
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-03-2009
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Aryl sulfamates were originally developed as inhibitors of steroid sulfatase, and have recently been shown to be powerful inactivators of a bacterial sulfatase, PaAtsA from Pseudomonas aeruginosa. We demonstrate that a simple aryl sulfamate, 3-nitrophenyl sulfamate, can inactivate sulfatases from various sources including snail, limpet and abalone. In each case inactivation was time-dependent and active-site directed, as demonstrated by protection against inactivation by substrate. These results suggest that such easily acquired aryl sulfamates can be used as reliable biochemical reagents for the study of sulfatases from a diverse array of sources.
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Synthesis and evaluation of dithiolethiones as novel cyclooxygenase inhibitors.
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-03-2009
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3H-1,2-Dithiole-3-thiones substituted with a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) or a 3,5-di-tert-butyl-4-methoxyphenyl group at the C5 position were prepared and their ability to inhibit the cyclooxygenase isoenzymes, COX-1 and COX-2 was evaluated. Both compounds were potent inhibitors of COX-2 (relative to rofecoxib), and while the phenol was a weak inhibitor of COX-1, the methyl ether gave no measurable inhibition. Docking studies of the two compounds into the COX-1 and -2 active sites showed that the methyl ether could only fit in the COX-2 active site whereas the phenol could be docked into both COX-1 and -2. This study reports a new mode for inhibitor binding to COX-1 and -2 and a novel structural scaffold for the development of COX-2 selective inhibitors.
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Synthesis of sulfated glucosaminides for profiling substrate specificities of sulfatases and fungal beta-N-acetylhexosaminidases.
Chembiochem
PUBLISHED: 01-22-2009
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Systematic sulfation: Sulfated glycoconjugates are degraded either by desulfation followed by glycoside cleavage, or by glycoside cleavage followed by desulfation. To study these processes, here we report the synthesis of four regioisomerically sulfated p-nitrophenyl glucosaminides from the common precursor p-nitrophenyl N-acetyl-beta-D-glucosaminide. These substrates allowed the rapid analysis of the substrate preferences of a set of four sulfatases and 24 hexosaminidases.Sulfated carbohydrates are components of many glycoconjugates, and are degraded by two major processes: cleavage of the sulfate ester by a sulfatase, or en bloc removal of a sulfated monosaccharide by a glycoside hydrolase. However, these processes have proved difficult to study owing to a lack of homogeneous, defined substrates. We describe here the synthesis of a series of p-nitrophenyl beta-D-glucosaminides bearing sulfate esters at the 2-, 3-, 4- or 6-positions, by divergent routes starting with p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The sulfated p-nitrophenyl beta-D-glucosaminides were used to study the substrate specificity of four sulfatases (from Helix pomatia, Patella vulgata, abalone, and Pseudomonas aeruginosa), and revealed significant differences in the preference of each of these enzymes for desulfation at different positions around the sugar ring. The 3-, 4- and 6-sulfated p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucosaminides were screened against a panel of 24 fungal beta-N-acetylhexosaminidases to assess their substrate specificity. While the 4- and 6-sulfates were substrates for many of the fungal enzymes investigated, only a single beta-N-acetylhexosaminidase, that from Penicillium chrysogenum, could hydrolyze the 3-sulfated p-nitrophenyl glycoside. Together these results demonstrate the utility of sulfated p-nitrophenyl beta-D-glucosaminides for the study of both sulfatases and glycoside hydrolases.
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The transgenic mouse assay as an alternative test method for regulatory carcinogenicity studies--implications for REACH.
Regul. Toxicol. Pharmacol.
PUBLISHED: 01-08-2009
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REACH, an EU regulation that requires the submission of safety data in support of the protection of human and environmental health, mandates that registration should be achieved with the minimum amount of animal testing possible. Under REACH, a two-year carcinogenicity assay may be required for certain chemicals produced at >1000 metric tonnes per year. In addition, some chemicals that are found to be genotoxic will also require testing. Alternative methods have been explored in an attempt to improve the predictivity of this bioassay as well as to reduce the number of animals used for such testing. This research has focused on the use of transgenic/knockout mouse models. Study results from selected models indicate that they are useful in hazard identification, even if they are not entirely suitable for risk assessment on their own. Carcinogenic hazard assessment can be greatly enhanced and animal use reduced if the traditional two-year rat bioassay is combined with a well conducted transgenic mouse assay. Importantly, the use of transgenic animals to supplement a traditional two-year carcinogenicity study may help reduce the number of false negatives, one of the unstated goals of REACH via the precautionary principle.
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A new anti-fibrotic drug attenuates cardiac remodeling and systolic dysfunction following experimental myocardial infarction.
Int. J. Cardiol.
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Pathological deposition of extracellular matrix in the non-infarct zone (NIZ) of the ventricle post myocardial infarction (MI) is a key contributor to cardiac remodeling and heart failure. FT011, a novel antifibrotic compound, was evaluated for its efficacy in neonatal cardiac fibroblasts (NCF) and in an experimental MI model.
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Sulfatase inhibitors: a patent review.
Expert Opin Ther Pat
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Steroid sulfatase (STS) converts sulfated hormones to free hormones of importance in hormone-dependent diseases such as breast cancer and endometriosis. Carbohydrate sulfatases degrade complex carbohydrates as part of normal cellular turnover; certain lysosomal storage disorders (LSDs) involve defective processing of sulfated glycosaminoglycans by mutant sulfatases.
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A purpose-synthesised anti-fibrotic agent attenuates experimental kidney diseases in the rat.
PLoS ONE
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Locally-active growth factors have been implicated in the pathogenesis of many diseases in which organ fibrosis is a characteristic feature. In the setting of chronic kidney disease (CKD), two such pro-fibrotic factors, transforming growth factor-? (TGF-?) and platelet-derived growth factor (PDGF) have emerged as lead potential targets for intervention. Given the incomplete organ protection afforded by blocking the actions of TGF-? or PDGF individually, we sought to determine whether an agent that inhibited the actions of both may have broader effects in ameliorating the key structural and functional abnormalities of CKD.
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Caterpillar labial saliva alters tomato plant gene expression.
J. Chem. Ecol.
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We examined the effects of Helicoverpa zea caterpillar labial saliva on tomato plant gene expression. Caterpillars with labial salivary glands (mock-ablated) and without (ablated) were fed on tomato plants for 24 hr; then, the leaf mRNA was analyzed with tomato microarrays. Analysis of the transcript profiles revealed 384 expressed sequence tags (ESTs) that were significantly altered due to herbivory compared to the non-wounded plants. The majority of the ESTs were quantitatively altered more so by mock-ablated caterpillars with labial salivary glands than ablated caterpillars. Particularly notable, ESTs encoding acid phosphatase, arginase, acidic endochitinase, dehydrin, polyphenol oxidase, protease inhibitors, and threonine deaminase were more highly stimulated by mock-ablated caterpillars than ablated caterpillars. In addition, tomato leaves were mechanically wounded with scissors and painted with labial salivary gland extract, autoclaved salivary gland extract, or water, and compared to non-wounded tomato plants. After 4 hr, these leaves were collected and a tomato microarray analysis of the mRNA revealed correlation of the gene expression of these leaves altered by mechanical wounding and painted with salivary gland extract to the gene expression of leaves fed on by mock-ablated caterpillars. We show that caterpillar labial saliva is an important component of herbivory that can alter plant gene expression.
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The reaction coordinate of a bacterial GH47 ?-mannosidase: a combined quantum mechanical and structural approach.
Angew. Chem. Int. Ed. Engl.
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Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting ?-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated ?-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.
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Conjugation of transferrin to azide-modified CdSe/ZnS core-shell quantum dots using cyclooctyne click chemistry.
Angew. Chem. Int. Ed. Engl.
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Twinkle twinkle quantum dot: Conjugation of biomolecules to azide-modified quantum dots (QDs) through a bifunctional linker, using strain-promoted azide-alkyne cycloaddition with the QD and a squaramide linkage to the biomolecule (see scheme). Transferrin-conjugated QDs were internalized by transferrin-receptor expressing HeLa cells.
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Localization of oleuropeyl glucose esters and a flavanone to secretory cavities of Myrtaceae.
PLoS ONE
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We report the widespread occurrence of structurally diverse oleuropeyl glucose esters, including the new diester eucaglobulin B, localized specifically to the essential oil secretory cavities of myrtaceous species. Clear taxonomic patterns in the composition of cavity extracts within the genus Eucalyptus are shown with species from subgenus Symphyomyrtus dominated by oleuropeyl glucose esters and species from subgenus Eucalyptus dominated instead by the flavanone, pinocembrin. We also examined the intra-species occurrence of oleuropeyl glucose esters by quantifying the abundant constituents cuniloside B and froggattiside A in trees from two populations of Eucalyptus polybractea R.T. Baker. All trees contained both compounds, which were positively correlated with total essential oil concentration. This apparent ubiquity of oleuropeyl glucose esters at both intra- and inter-specific levels in Eucalyptus is indicative of important physiological or ecological functions. The significance of their prevalence and the sequestration of these esters and also pinocembrin to the extracellular domain of secretory cavities is discussed in light of their potential biological activities and our findings that they are spatially segregated to the exterior of cavity lumina. The localization of oleuropeyl glucose esters to a specific and isolatable tissue type has the potential to aid in future elucidation of function and biosynthesis.
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Synthesis of glycosyl fluorides from thio-, seleno-, and telluroglycosides and glycosyl sulfoxides using aminodifluorosulfinium tetrafluoroborates.
Carbohydr. Res.
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Glycosyl fluorides can be synthesized from thio-, seleno-, and telluroglycosides and glycosyl sulfoxides using the aminodifluorosulfinium tetrafluoroborate reagents Xtalfluor-E and -M, with or without added N-bromosuccinimide. Mechanistic studies provide evidence that fluoride is delivered from the tetrafluoroborate counterion.
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FT23, an orally active antifibrotic compound, attenuates structural and functional abnormalities in an experimental model of diabetic cardiomyopathy.
Clin. Exp. Pharmacol. Physiol.
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Diabetic cardiomyopathy is characterized by early diastolic dysfunction and structural changes, such as interstitial fibrosis and cardiac hypertrophy. Using the Ren-2 rat model, we sought to investigate the effect of FT23 on the structural and functional changes associated with diabetic cardiomyopathy. Heterozygous Ren-2 rats were rendered diabetic with streptozotocin by tail vein injection. Rats were then treated with FT23 (200 mg/kg per day by gavage twice daily) or vehicle from Week 8 to Week 16 after the onset of diabetes. Echocardiography was performed to assess heart function before the rats were killed and their hearts collected for histological and molecular biological assessment. The antifibrotic effect of FT23 was compared with that of tranilast in neonatal cardiac fibroblasts when stimulated with transforming growth factor (TGF)-? (5 ng/mL) at 30, 50 and 100 umol/L. FT23 exhibited greater inhibition of TGF-?-induced collagen production in neonatal cardiac fibroblasts, as measured by a [(3) H]-proline incorporation assay, compared with its parental compound tranilast. In the in vivo study, FT23 significantly attenuated the increased heart weight : bodyweight ratio in FT23-treated diabetic Ren-2 rats. Diastolic dysfunction, as measured by mitral valve (MV) E/A ratio and MV deceleration time, was also significantly attenuated by FT23. Picrosirius red-stained heart sections revealed that cardiac fibrosis in the diabetic rats was reduced by FT23 compared with that in vehicle-treated rats, with a concomitant reduction in collagen I immunostaining and infiltration of macrophages, as demonstrated by ED1 immunostaining. The results of the present study suggest that FT23 inhibits the activity of TGF-? and attenuates structural and functional manifestations of diastolic dysfunction observed in a model of diabetic cardiomyopathy.
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FT011, a new anti-fibrotic drug, attenuates fibrosis and chronic heart failure in experimental diabetic cardiomyopathy.
Eur. J. Heart Fail.
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Cardiac remodelling in diabetes includes pathological accumulation of extracellular matrix and myocyte hypertrophy that contribute to heart dysfunction. Attenuation of remodelling represents a potential therapeutic target. We tested this hypothesis using a new anti-fibrotic drug, FT011 (Fibrotech Therapeutics Pty Ltd), on diabetic Ren-2 rats, a model which replicates many of the structural and functional manifestations of diabetic cardiomyopathy in humans.
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Discovery of inhibitors of Leishmania ?-1,2-mannosyltransferases using a click-chemistry-derived guanosine monophosphate library.
PLoS ONE
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Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of ?-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose-dependent ?-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl ?-D-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen ?-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism.
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2-Morpholinoisoflav-3-enes as flexible intermediates in the synthesis of phenoxodiol, isophenoxodiol, equol and analogues: vasorelaxant properties, estrogen receptor binding and Rho/RhoA kinase pathway inhibition.
Bioorg. Med. Chem.
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Isoflavone consumption correlates with reduced rates of cardiovascular disease. Epidemiological studies and clinical data provide evidence that isoflavone metabolites, such as the isoflavan equol, contribute to these beneficial effects. In this study we developed a new route to isoflavans and isoflavenes via 2-morpholinoisoflavenes derived from a condensation reaction of phenylacetaldehydes, salicylaldehydes and morpholine. We report the synthesis of the isoflavans equol and deoxygenated analogues, and the isoflavenes 7,4-dihydroxyisoflav-3-ene (phenoxodiol, haganin E) and 7,4-dihydroxyisoflav-2-ene (isophenoxodiol). Vascular pharmacology studies reveal that all oxygenated isoflavans and isoflavenes can attenuate phenylephrine-induced vasoconstriction, which was unaffected by the estrogen receptor antagonist ICI 182,780. Furthermore, the compounds inhibited U46619 (a thromboxane A(2) analogue) induced vasoconstriction in endothelium-denuded rat aortae, and reduced the formation of GTP RhoA, with the effects being greatest for equol and phenoxodiol. Ligand displacement studies of rat uterine cytosol estrogen receptor revealed the compounds to be generally weak binders. These data are consistent with the vasorelaxation activity of equol and phenoxodiol deriving at least in part by inhibition of the RhoA/Rho-kinase pathway, and along with the limited estrogen receptor affinity supports a role for equol and phenoxodiol as useful agents for maintaining cardiovascular function with limited estrogenic effects.
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NF-?B and matrix-dependent regulation of osteopontin promoter activity in allylamine-activated vascular smooth muscle cells.
Oxid Med Cell Longev
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Repeated cycles of oxidative injury by allylamine in vivo induce a proliferative rat vascular (aortic) smooth muscle cell (vSMC) phenotype characterized by matrix-dependent enhancement of mitogenic sensitivity, changes in cell surface integrin expression, and osteopontin (opn) overexpression. Here, we show that constitutive and mitogen-stimulated NF-?B DNA binding activity is enhanced in allylamine vSMCs. Matrix-specific changes in cellular Rel protein expression were observed in allylamine vSMCs. The NF-?B DNA binding element located at -1943 in the 5-UTR strongly inhibited opn promoter activity in allylamine vSMCs, and this response was regulated by the extracellular matrix. Constitutive increases in opn promoter activity were only seen when allylamine cells were seeded on a fibronectin substrate, and this response was independent of the NF-?B DNA binding sequence within the regulatory region. Thus, NF-?B functions as a critical regulator of the allylamine-induced proliferative phenotype in vSMCs.
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Coal-tar-based pavement sealcoat and PAHs: implications for the environment, human health, and stormwater management.
Environ. Sci. Technol.
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Coal-tar-based sealcoat products, widely used in the central and eastern U.S. on parking lots, driveways, and even playgrounds, are typically 20-35% coal-tar pitch, a known human carcinogen that contains about 200 polycyclic aromatic hydrocarbon (PAH) compounds. Research continues to identify environmental compartments-including stormwater runoff, lake sediment, soil, house dust, and most recently, air-contaminated by PAHs from coal-tar-based sealcoat and to demonstrate potential risks to biological communities and human health. In many cases, the levels of contamination associated with sealed pavement are striking relative to levels near unsealed pavement: PAH concentrations in air over pavement with freshly applied coal-tar-based sealcoat, for example, were hundreds to thousands of times higher than those in air over unsealed pavement. Even a small amount of sealcoated pavement can be the dominant source of PAHs to sediment in stormwater-retention ponds; proper disposal of such PAH-contaminated sediment can be extremely costly. Several local governments, the District of Columbia, and the State of Washington have banned use of these products, and several national and regional hardware and home-improvement retailers have voluntarily ceased selling them.
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Structural and mechanistic insight into N-glycan processing by endo-?-mannosidase.
Proc. Natl. Acad. Sci. U.S.A.
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N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-?-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-?-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (?/?)(8) barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-Man. Using the bespoke substrate ?-Glc-1,3-?-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-?-mannosidase inhibitor ?-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, ?-Glc-1,3-isofagomine, and with the reducing-end product ?-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.