Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Organic-inorganic poly(methyl methacrylate) hybrids with confined polyhedral oligosilsesquioxane macromonomers

We herein report the synthesis of organic-inorganic hybrid poly(methyl methacrylate) containing 1 polyhedral oligosilsesquioxanes. Octakis(3-hydroxypropyldimethylsiloxy)octasilsesquioxane (OHPS) was synthesized from octakis(hydridodimethylsiloxy)octasilsesquioxane [Si8O12(OSiMe2H)(8), Q(8)M(8)(H)] following literature procedures. Octakis(tnethacryloxypropyldimethylsiloxy) octasilsesquioxane (OMPS) was synthesized via the reaction of methacryloyl chloride or methacrylic acid anhydride with OHPS, with the latter giving improved purity. Polymerization of OMPS with methyl inethacrylate using a dibenzoylperoxide initiator gave a highly cross-linked polymer. Characterization of the polymer was performed using Fourier transform IR spectroscopy, Si-29 NMR, differential scanning calorimetry, thermogravimetric analysis, atomic force microscopy, and transmission electron microscopy with energy-dispersive X-ray analysis. The polymer was found to be largely homogeneous. Increasing the OMPS concentration in the polymer gave increased decomposition and glass transition temperatures.

Facile synthesis of rhodamine esters using acetyl chloride in alcohol solution

The rhodamines are a highly fluorescent class of compound used in many different fields of research, from the lasing medium in dye lasers to biological stains and markers for cellular drug resistance. In this study, esters (2-7) of rhodamine 110 (1) were conveniently prepared via the addition of acetyl chloride to a solution of the free acid (1) in the appropriate alcohol. This method conferred several advantages over previous preparations, namely that for low boiling alcohols, simple evaporation of the solution afforded the ester in quantitative yield with no need for purification. For higher boiling point alcohols, a method has been developed which allows the separation of longer chain esters from the alcohol solvent.

Macrophage-specific expression of human lysosomal acid lipase corrects inflammation and pathogenic phenotypes in lal(-/-) mice

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in the cell. The downstream metabolites of these compounds serve as hormonal ligands for nuclear receptors and transcription factors. Genetic ablation of the lal gene in the mouse caused malformation of macrophages and inflammation-triggered multiple pathogenic phenotypes in multiple organs. To assess the relationship between macro phages and lal(-/-) pathogenic phenotypes, a macrophage-specific doxycycline-inducible transgenic system was generated to induce human LAL (hLAL) expression in the lal(-/-) genetic background under control of the 7.2-kb c-fins promoter/intron2 regulatory sequence. Doxycycline-induced hLAL expression in macrophages significantly ameliorated aberrant gene expression, inflammatory cell (neutrophil) influx, and pathogenesis in multiple organs. These studies strongly support that neutral lipid metabolism in macrophages contributes to organ inflammation and pathogenesis.

Acibenzolar-S-methyl and methyl jasmonate treatments of glasshouse-grown freesias suppress post-harvest petal specking caused by Botrytis cinerea

Compounds that activate host plant defence responses potentially offer socio-environmentally sound alternative methods for disease control. In a series of glasshouse trials over 2 years, pre-harvest sprays with acibenzolar-S-methyl (ASM) and methyl jasmonate (MeJA) were tested for suppression of post-harvest infection of cut Freesia hybrida L. flowers by Botrytis cinerea. For the ASM treatments, variability in reducing the incidence of B. cinerea disease was observed between years freesia varieties, incubation temperatures and ASM concentrations. In the first year, the greatest reductions in lesion numbers on ASM-treated var. 'Cote d'Azur' were recorded using 2.86 mM ASM. For three different post-harvest temperature regimes, the relative reductions in lesion numbers, compared to untreated controls, were 45% at 5 degrees C, 40% at 12 degrees C and 30% at 20 degrees C, respectively. In the second year, lesion numbers were most reduced using 1.43 mM ASM to treat freesia var. 'Dukaat' flowers. Here, the relative reductions were to 44% at 5 degrees C, 26% at 12 degrees C and 51% at 20 degrees C. MeJA treatments were, in general, more consistently effective than ASM treatments in reducing lesion numbers and lesion diameters on cut freesia flowers. MeJA-treated (0.2 mM) freesia flowers (var. 'Dukaat') incubated at 20 degrees C showed relative reductions of 62%, and 45% for lesion number and lesion diameter, respectively. The differing efficacy between ASM and MeJA treatments could be attributed to their differential abilities to induce the salicylic acid (SA)-mediated vs. the jasmonic acid (JA)-mediated host defence pathways, respectively.

A blank slate? Layer-by-layer deposition of hyaluronic acid and chitosan onto various surfaces

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called blank slate: essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum

The necrotrophic fungal pathogen Fusarium pseudograminearum (F. pseudograminearum) causes crown rot disease (CR) in wheat. This host-pathogen interaction has not been studied previously at the molecular level. In this study. using real-time quantitative PCR, the expression of 26 selected wheat genes was examined 1, 2 and 4 days after inoculation of wheat seedlings of the CR susceptible cultivar Kennedy and the partially field-resistant cultivar Sunco. Reproducible induction of eight defence genes consisting of PR1.1, PR2 (beta,1-3 glucanase), PR3 (chitinase), PR4 (wheativin), PR5 (thaumatin-like protein). TaPERO (peroxidase), PR10 and TaGLP2a (germin-like) was observed. These genes were induced in both cultivars, however. some genes were induced more rapidly in Sunco than in Kennedy. MJ treatment also induced the above pathogen responsive defence genes in both cultivars while benzo(1,2,3)thiadiazole-7-carbothionic acid S-methyl ester (BTH) treatment weakly induced them in Kennedy only. Similarly. treatment with MJ before inoculation significantly delayed the development of necrotic symptoms for 2 weeks in both wheat cultivars, while BTH pre-treatments delayed symptom development in Kennedy only. The chemically induced protection, therefore, correlated with induction of the F. pseudograminearum-responsive genes. These results support the emerging role of jasmonate signalling in defence against necrotrophic fungal pathogens in monocots and future manipulation of this pathway may improve CR resistance in wheat. (c) 2006 Elsevier Ltd. All rights reserved.

Purification of phosphoric acid by solvent extraction

This work follows a feasibility study (187) which suggested that a process for purifying wet-process phosphoric acid by solvent extraction should be economically viable. The work was divided into two main areas, (i) chemical and physical measurements on the three-phase system, with or without impurities; (ii) process simulation and optimization. The object was to test the process technically and economically and to optimise the type of solvent. The chemical equilibria and distribution curves for the system water - phosphoric acid - solvent for the solvents n-amyl alcohol, tri-n-butyl phosphate, di-isopropyl ether and methyl isobutyl ketone have been determined. Both pure phosphoric acid and acid containing known amounts of naturally occurring impurities (Fe P0 4 , A1P0 4 , Ca3(P04)Z and Mg 3(P0 4 )Z) were examined. The hydrodynamic characteristics of the systems were also studied. The experimental results obtained for drop size distribution were compared with those obtainable from Hinze's equation (32) and it was found that they deviated by an amount related to the turbulence. A comprehensive literature survey on the purification of wet-process phosphoric acid by organic solvents has been made. The literature regarding solvent extraction fundamentals and equipment and optimization methods for the envisaged process was also reviewed. A modified form of the Kremser-Brown and Souders equation to calculate the number of contact stages was derived. The modification takes into account the special nature of phosphoric acid distribution curves in the studied systems. The process flow-sheet was developed and simulated. Powell's direct search optimization method was selected in conjunction with the linear search algorithm of Davies, Swann and Campey. The objective function was defined as the total annual manufacturing cost and the program was employed to find the optimum operating conditions for anyone of the chosen solvents. The final results demonstrated the following order of feasibility to purify wet-process acid: di-isopropyl ether, methylisobutyl ketone, n-amyl alcohol and tri-n-butyl phosphate.

Subthalamic nucleus neurones in slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice show irregular, dopamine-reversible firing pattern changes, but without synchronous activity

The loss of dopamine in idiopathic or animal models of Parkinson's disease induces synchronized low-frequency oscillatory burst-firing in subthalamic nucleus neurones. We sought to establish whether these firing patterns observed in vivo were preserved in slices taken from dopamine-depleted animals, thus establishing a role for the isolated subthalamic-globus pallidus complex in generating the pathological activity. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed significant reductions of over 90% in levels of dopamine as measured in striatum by high pressure liquid chromatography. Likewise, significant reductions in tyrosine hydroxylase immunostaining within the striatum (>90%) and tyrosine hydroxylase positive cell numbers (65%) in substantia nigra were observed. Compared with slices from intact mice, neurones in slices from MPTP-lesioned mice fired significantly more slowly (mean rate of 4.2 Hz, cf. 7.2 Hz in control) and more irregularly (mean coefficient of variation of inter-spike interval of 94.4%, cf. 37.9% in control). Application of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) and the GABAA receptor antagonist picrotoxin caused no change in firing pattern. Bath application of dopamine significantly increased cell firing rate and regularized the pattern of activity in cells from slices from both MPTP-treated and control animals. Although the absolute change was more modest in control slices, the maximum dopamine effect in the two groups was comparable. Indeed, when taking into account the basal firing rate, no differences in the sensitivity to dopamine were observed between these two cohorts. Furthermore, pairs of subthalamic nucleus cells showed no correlated activity in slices from either control (21 pairs) or MPTP-treated animals (20 pairs). These results indicate that the isolated but interconnected subthalamic-globus pallidus network is not itself sufficient to generate the aberrant firing patterns in dopamine-depleted animals. More likely, inputs from other regions, such as the cortex, are needed to generate pathological oscillatory activity. © 2006 IBRO.

Enzyme catalysed modification of polymers

The aim of this project was to investigate the enzyme catalysed modification of synthetic polymers. It was found that an immobilised lipase from Candida antartica (Novozyme 435) catalysed the selective epoxidation of poly(butadiene) in the presence of hydrogen peroxide and catalytic quantities of acetic acid. The cis and trans double bonds of the backbone were epoxidised in yields of up to 60 % whilst the pendent vinyl groups were untouched. The effect of varying a number of reaction parameters was investigated. These studies suggested that higher yields of epoxide could not be obtained because of the conformational properties of the partially epoxidised polymer. Application of this process to the Baeyer-Villiger reaction of poly(vinyl phenyl ketone) and poly(vinyl methyl ketone) were unsuccessful. The lack of reactivity was found to be a property of the polymer rather than the enzymatic system employed. Attempts to modify hydroxyl containing polymers and polymers bearing active esters close to the polymer backbone were unsuccessful. Steric factors appear to be the most important influence on the outcome of the reactions.

Cyclodextrin complexes of antimicrobial agents

Poorly water-soluble drugs show an increase in solubility in the presence of cyclodextrins (CyD) due to the formation of a water-soluble complex between the drug and dissolved CyD. This study investigated the interactions of -Cyd and hydroxypropyl--CyD (HP--CyD, M.S. = 0.6) with antimicrobial agents of limited solubility in an attempt to increase their microbiological efficacy. The agents studied were chlorhexidine dihydrochloride (CHX), p-hydroxybenzoic acid esters (methyl, ethyl, proply and butyl) and triclosan. The interactions between the antimicrobials and CyDs were studied in solution and solid phases. Phase solubility studied revealed an enhancement in the aqueous drug solubility in the presence of the CyD and also gave an indication of the complex stability constant (Ks). The temperature-dependence of the stability constant of the complex was modelled by the van't Hoff plot which yielded the thermodynamic parameters for complexation. Further confirmation of the inclusion of the antimicrobials within the cavity of the CyDs in aqueous solution was obtained from proton magnetic resonance and ultraviolet absorption spectroscopies. The former method indicated that the chlorophenyl moiety of the CHX was included within the -CyD cavity and the stoichiometry of the complex formed was 1:1. The solid-phase complexes were prepared by freeze-drying. The inclusion complex of triclosan with HP--CyD was obtained from aqueous solution with the addition of ammonia. Evidence to confirm complex formation was obtained from DSC, IR and X-ray powder diffraction studies. Dissolution studies of the solid inclusion complexes using the dispersed powder technique illustrated their superior solubilities as compared to the equimolar physical mix of the guest and CyD. It was shown that these solutions of the complex were supersaturated with respect to the free guest. This was further demonstrated by diffusion studies which showed the flux of free drug from donor solutions of the antimicrobial-CyD complex to be significantly greater than the flux from donor suspensions of drug alone.

Bifunctional organorhodium solid acid catalysts for methanol carbonylation

Robust, bifunctional catalysts comprising Rh(CO)(Xantphos) exchanged phosphotungstic acids of general formulas [Rh(CO)(Xantphos)]+n[H3–nPW12O40]n− have been synthesized over silica supports which exhibit tunable activity and selectivity toward direct vapor phase methanol carbonylation. The optimal Rh:acid ratio = 0.5, with higher rhodium concentrations increasing the selectivity to methyl acetate over dimethyl ether at the expense of lower acidity and poor activity. On-stream deactivation above 200 °C reflects Rh decomplexation and reduction to Rh metal, in conjunction with catalyst dehydration and loss of solid acidity because of undesired methyl acetate hydrolysis, but can be alleviated by water addition and lower temperature operation.

Mechanistic studies on the degradation of gasoline oxygenates by advanced oxidation technologies and the reaction of 4-methyl-1,2,4-triazoline-3,5-dione with tetracyclopropylethylene

Gasoline oxygenates (MTBE, methyl tert-butyl ether; DIPE, di-isopropyl ether; ETBE, ethyl tert-butyl ether; TAME, tert-amyl ether) are added to gasoline to boost octane and enhance combustion. The combination of large scale use, high water solubility and only minor biodegradability has now resulted in a significant gasoline oxygenate contamination occurring in surface, ground, and drinking water systems. Combination of hydroxyl radical formation and the pyrolytic environment generated by ultrasonic irradiation (665 kHz) leads to the rapid degradation of MTBE and other gasoline oxygenates in aqueous media. ^ The presence of oxygen promotes the degradation processes by rapid reaction with carbon centered radicals indicating radical processes involving O 2 are significant pathways. A number of the oxidation products were identified. The formation of products (alcohols, ketones, aldehydes, esters, peroxides, etc) could be rationalized by mechanisms which involve hydrogen abstraction by OH radical and/or pyrolysis to form carboncentered radicals which react with oxygen and follow standard oxidation chain processes. ^ The reactions of N-substituted R-triazolinediones (RTAD; R = CH 3 or phenyl) have attracted considerable interest because they exhibit a number of unusual mechanistic characteristics that are analogous to the reactions of singlet oxygen (1O2) and offer an easy way to provide C-N bond(s) formation. The reactions of triazolinedione with olefins have been widely studied and aziridinium imides are generally accepted to be the reactive intermediates. ^ We observed the rapid formation of an unusual intermediate upon mixing tetracyclopropylethylene with 4-methyl-1,2,4-triazoline-3,5-dione in CDCl 3. Detailed characterization by NMR (proton, 13C, 2-D NMRs) indicates the intermediate is 5,5,6,6-tetracyclopropyl-3-methyl-5,6-dihydro-oxazolo[3,2- b][1,2,4]-triazolium-2-olate. Such products are extremely rare and have not been studied. Upon warming the intermediate is converted to 2 + 2 diazetidine (major) and ene product (minor). ^ To further explore the kinetics and dynamics of the reaction activation energies were obtained using Arrhenius plots. Activation energies for the formation of the intermediate from reactants, and 2+2 adduct from the intermediate were determined as 7.48 kcal moll and 19.8 kcal mol−1 with their pre-exponential values of 2.24 × 105 dm 3 mol−1 sec−1 and 2.75 × 108 sec−1, respectively, meaning net slow reactions because of low pre-exponential values caused by steric hindrance. ^

Syntheses of aza analogues of kainic acid

The kainate receptors are one of the three major groups of ionotropic glutamate receptors in the mammalian central nervous system. They are so named after their most potent agonist, kainic acid (KA), a natural product isolated from the seaweed Diginea simplex. This compound shows both neuroexcitatory and excitotoxic activities, and is an important pharmacological tool for neurophysiological studies. We predict that the more synthetically accessible aza analogues of kainic acid, could act as functional mimics of KA. These could be produced by the 1,3-dipolar cycloaddition of diazoalkanes with trans glutaconate esters. ^ 1,3-Dipolar cycloadditions have been shown to produce 1-pyrazolines that isomerize into 2-pyrazolines. The 1- and 2-pyrazolines can be precursors to aza analogs of kainoids. The regioselectivity, relative stereochemistry and isomerization of the 1-pyrazolines into 2-pyrazolines have been evaluated. Reductions of the 1- and 2-pyrazolines produced aza analogs of kainoids. TMS diazomethane was used as the dipole in 1,3-dipolar cycloaddition reactions leading to aza KA analogs via 2-pyrazolines. A systematic study of cycloaddition-isomerization processes involving TMS-diazomethane and various α, β-unsaturated dipolarophiles has been undertaken. 1H-NMR monitoring of the reaction mixture compositions during the cycloaddition reaction revealed evidence of retro-dipolar cycloaddition processes. Faster formation of 4,5- trans-1-pyrazoline at the beginning of the reaction and subsequent isomerization of this product into 4,5-cis-1-pyrazoline via a retro-dipolar cycloaddition has been observed. Increased reaction time and/or reaction temperature preferentially caused the irreversible isomerization of 4,5-cis-1-pyrazoline into 4,5-cis-2-pyrazoline, which led to high yields of 4,5-cis-2-pyrazolines in the overall process. ^ Two syntheses of the 5-unsubstituted aza-kainic acid have been performed; first, via the reduction of the TMS-eliminated 2-pyrazoline from TMS diazomethane; second by the direct reduction of 1-pyrazoline with Hg/Al-amalgam. 5-Phenyl aza-kainic acid has been produced by direct reduction of 1-pyrazoline, obtained in the reaction of phenyldiazomethane and dibenzyl glutaconate, with Hg/Al-amalgam. ^ Current responses to aza kainate analogs in Aplysia whole cell buccal ganglia indicate potent neuroexcitatory activity. The repetitive exposure of neuronal cells to the 5-unsubstituted aza-kainic acid led to non-desensitizing current responses, showing both binding affinity and neuronal ion-channel activation by the synthesized agonist compound. ^

Total phospholipid fatty acid concentrations structrures of ODP Hole 191-1179 samples (Table 1)

Sediment samples ranging from 0.05 to 278 m below sea floor (mbsf) at a Northwest Pacific deep-water (5564 mbsl) site (ODP Leg 191, Site 1179) were analyzed for phospholipid fatty acids (PLFAs). Total PLFA concentrations decreased by a factor of three over the first meter of sediment and then decreased at a slower rate to approximately 30 mbsf. The sharp decrease over the first meter corresponds to the depth of nitrate and Mn(IV) reduction as indicated by pore water chemistry. PLFA-based cell numbers at site 1179 had a similar depth profile as that for Acridine orange direct cell counts previously made on ODP site 1149 sediments which have a similar water depth and lithology. The mole percentage of straight chain saturated PLFAs increases with depth, with a large shift between the 0.95 and 3.95 mbsf samples. PLFA stable carbon isotope ratios were determined for sediments from 0.05 to 4.53 mbsf and showed a general trend toward more depleted d13C values with depth. Both of these observations may indicate a shift in the bacterial community with depth across the different redox zones inferred from pore water chemistry data. The PLFA 10me16:0, which has been attributed to the bacterial genera Desulfobacter in many marine sediments, showed the greatest isotopic depletion, decreasing from -20 to -35 per mil over the first meter of sediment. Pore water chemistry suggested that sulfate reduction was absent or minimal over this same sediment interval. However, 10me16:0 has been shown to be produced by recently discovered anaerobic ammonium oxidizing (anammox) bacteria which are known chemoautotrophs. The increasing depletion in d13C of 10me16:0 with the unusually lower concentration of ammonium and linear decrease of nitrate concentration is consistent with a scenario of anammox bacteria mediating the oxidation of ammonium via nitrite, an intermediate of nitrate reduction.