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Model Substrate (model + substrate)
Selected Abstracts"Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction productsELECTROPHORESIS, Issue 13 2009Helene Piccard Abstract A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest. [source] A Density Functional Study of the Hydrogenation of Ketones Catalysed by Neutral Rhodium-Diphosphane ComplexesEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 21 2006Francine Agbossou-Niedercorn Abstract The potential energy profile of RhI -catalysed hydrogenation of ketones has been computed for the simple model system [Rh{H3POCH2CH2N(H)PH3}(Cl)] using DFT calculations. The general sequence of the catalytic cycle involves coordination of the carbonyl derivative to the neutral RhI complex followed by oxidative addition of molecular hydrogen providing rhodium dihydride intermediates. The latter are converted into alkoxy hydrides by a migratory insertion reaction. Reductive elimination of the alcohol and substitution of the latter by the incoming substrate completes the catalyticcycle. Intermediates and transition states of all catalyticsteps have been located. Two isomeric derivatives bearingthe model substrate have been found for the [Rh{H3POCH2CH2N(H)PH3}(Cl)(H2CO)] complex. Eight diastereomeric pathways have been followed for the cis addition of molecular hydrogen to [Rh{H3POCH2CH2N(H)PH3}(Cl)(H2CO)] leading to eight distinct isomeric dihydride intermediates. Four dihydride complexes can be considered as the more accessible compounds. The site preference for migratory insertion and transition states discriminates the main path of the catalytic reaction. Migratory insertion to form the alkoxy hydride constitute the turn over limiting step of the process. The potential energy profile has been found to be smooth without excessive activation barriers. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37RvFEBS JOURNAL, Issue 1 2009Md. Suhail Alam The whiB -like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV,Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe,2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe,2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe,2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe,2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis. [source] Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cellsFEBS JOURNAL, Issue 24 2001Antonios Klokouzas Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter. This paper shows that transport of the model substrate, 3 µm dinitrophenyl S -glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2,20 µm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action. [source] Mutations towards enantioselectivity adversely affect secretion of Pseudomonas aeruginosa lipaseFEMS MICROBIOLOGY LETTERS, Issue 1 2008Sascha Hausmann Abstract Lipases are important biocatalysts used as detergent additives to manufacture biodiesel, and in particular, for the production of enantiopure compounds such as alcohols, amines and carboxylic acids. Extensive efforts were conducted trying to optimize lipase properties and lipase LipA of Pseudomonas aeruginosa comprises the best-studied example in terms of optimizing enantioselectivity by application of numerous directed evolution methods. Its enantioselectivity in the asymmetric hydrolysis of the model substrate 2-methyldecanoic acid p -nitrophenyl ester was increased from E=1.1 for the wild-type enzyme to E=51 for the best (S)-enantioselective variant which carried six amino acid exchanges. We have observed that overexpression of this variant in the homologous host resulted in only marginal yields of enzyme in the bacterial culture supernatant, suggesting that the enantioselective LipA variant was secreted with only low efficiency. Hence, we have analysed the secretion of this lipase variant and compared it to variants carrying either the respective single mutations or some combinations. We report here the identification of two amino acid substitutions located on the protein surface, which significantly impair lipase secretion. [source] Accurate substrate modelling of RF CMOSINTERNATIONAL JOURNAL OF NUMERICAL MODELLING: ELECTRONIC NETWORKS, DEVICES AND FIELDS, Issue 3 2006M. S. Alam Abstract The losses within the substrate of an RF IC can have significant effect on performance in a mixed signal application. In order to model substrate coupling accurately, it is represented by an RC network to account for both resistive and dielectric losses at high frequency (> 1 GHz). A small-signal equivalent circuit model of an RF IC inclusive of substrate parasitic effect is analysed in terms of its y -parameters and an extraction procedure for substrate parameters has been developed. By coupling the extracted substrate parameters along with extrinsic resistances associated with gate, source and drain, a standard BSIM3 model has been extended for RF applications. The new model exhibits a significant improvement in prediction of output reflection coefficient S22 in the frequency range from 1 to 10 GHz in device mode of operation and for a low noise amplifier (LNA) at 2.4 GHz. Copyright © 2006 John Wiley & Sons, Ltd. [source] Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation MutagenesisADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 18 2009Despina Abstract Directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to the old yellow enzyme homologue YqjM in the quest to broaden its substrate scope, while controlling the enantioselectivity in the bioreduction of a set of substituted cyclopentenone and cyclohexenone derivatives. Guided by the known crystal structure of YqjM, 20 residues were selected as sites for saturation mutagenesis, a pooling strategy based on the method of Phizicky [M. R. Martzen, S. M. McCraith, S. L. Spinelli, F. M. Torres, S. Fields, E. J. Grayhack, E. M. Phizicky, Science1999, 286, 1153,1155] being used in the GC screening process. The genes of some of the hits were subsequently employed as templates for randomization experiments at the other putative hot spots. Both (R)- and (S)-selective variants were evolved using 3-methylcyclohexenone as the model substrate in the asymmetric bioreduction of the olefinic functionality, only small mutant libraries and thus minimal screening effort being necessary. Some of the best mutants also proved to be excellent catalysts when testing other prochiral substrates without resorting to additional mutagenesis/screening experiments. Thus, the results constitute an important step forward in generalizing the utility of ISM as an efficient method in laboratory evolution of enzymes as catalysts in organic chemistry. [source] Metal-Free and Copper-Promoted Single-Pot Hydrocarboxylation of Cycloalkanes to Carboxylic Acids in Aqueous MediumADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 17 2009Marina Abstract A simple and effective method for the transformation, under mild conditions and in aqueous medium, of various cycloalkanes (cyclopentane, cyclohexane, methylcyclohexane, cis - and trans -1,2-dimethylcyclohexane, cycloheptane, cyclooctane and adamantane) into the corresponding cycloalkanecarboxylic acids bearing one more carbon atom, is achieved. This method is characterized by a single-pot, low-temperature hydrocarboxylation reaction of the cycloalkane with carbon monoxide, water and potassium peroxodisulfate in water/acetonitrile medium, proceeding either in the absence or in the presence of a metal promoter. The influence of various reaction parameters, such as type and amount of metal promoter, solvent composition, temperature, time, carbon monoxide pressure, oxidant and cycloalkane, is investigated, leading to an optimization of the cyclohexane and cyclopentane carboxylations. The highest efficiency is observed in the systems promoted by a tetracopper(II) triethanolaminate-derived complex, which also shows different bond and stereoselectivity parameters (compared to the metal-free systems) in the carboxylations of methylcyclohexane and stereoisomeric 1,2-dimethylcyclohexanes. A free radical mechanism is proposed for the carboxylation of cyclohexane as a model substrate, involving the formation of an acyl radical, its oxidation and consequent hydroxylation by water. Relevant features of the present hydrocarboxylation method, besides the operation in aqueous medium, include the exceptional metal-free and acid-solvent-free reaction conditions, a rare hydroxylating role of water, substrate versatility, low temperatures (ca. 50,°C) and a rather high efficiency (up to 72% carboxylic acid yields based on cycloalkane). [source] Prevention of Manganese Precipitation during the Continuous Selective Partial Oxidation of Methyl Aromatics with Molecular Oxygen in Supercritical WaterADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2009Joan Fraga-Dubreuil Abstract Presented here is an investigation of the activity and recovery of the homogeneous manganese/bromide catalyst during the continuous flow oxidation of o -xylene, as model substrate, with molecular oxygen (O2) in supercritical water (scH2O). Two strategies are discussed for preventing catalyst precipitation, mainly in the form of oxides such as manganese(IV) oxide, The first strategy involves varying the manganese:bromide ratio using either manganese(II) acetate or manganese(II) bromide in the presence of hydrobromic and other acids. The results show that the effect of acidity and bromide concentration plays an important role in preventing the manganese/bromide catalyst from precipitating. The second strategy uses aromatic carboxylic acids in combination with the manganese/bromide catalyst, particularly benzoic acid, which improves the catalyst recovery dramatically over a certain range of acid concentrations. Our studies show how the presence of an organic acid and/or its precursors is important in stabilising the catalyst. Our results are rationalised on the basis of a tentative reaction mechanism. [source] Adhesion of Staphylococcus aureus and Staphylococcus epidermidis to the Episkin® reconstructed epidermis model and to an inert 304 stainless steel substrateJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004G. Lerebour Abstract Aims:, The aim of this study was to evaluate the respective influence of the physicochemical interactions and the roughness involved in the first part of the biological substrate biocontamination. Methods and Results:, Therefore we compared the bioadhesion results obtained on the biological model substrate (Episkin®) and on a commonly employed inert substrate (AISI 304 stainless steel), frequently used either in dermatology or in development of medical devices. The two studied strains presented different characteristics, both physicochemical and microbiological. Staphylococcus epidermidis, a relatively hydrophobic bacteria capable of exchanging interactions which are principally of the van der Waals type, adhered more to 304 steel than to the surface of reconstituted skin. As for S. aureus, an essentially basic, hydrophilic bacteria, was more adherent to Episkin® (a bipolar, hydrophilic substrate) than to stainless steel (a unipolar, basic, hydrophilic substrate). Conclusions:, In the absence of electrostatic interactions, the adhesion of substrate-dependent bacteria to the surface of reconstituted skin was dependent upon the balance between ,LW, ,+ and ,,. Significance and Impact of the Study:, Consequently, so as to restrict microbial adhesion and reduce adhesive binding between micro-organisms and the surface of the skin, it would be preferable to render this substrate hydrophobic and apolar through the use of appropriate surface treatment. [source] Kinetic behaviour of the adsorption and photocatalytic degradation of salicylic acid in aqueous TiO2 microsphere suspensionJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2004XZ Li Abstract A new photocatalyst, named TiO2 microspheres, prepared by a sol-spraying-calcination method, can freely suspend with air bubbling in its aqueous suspension and easily settle down from a water phase under gravity. The experimental results demonstrated that TiO2 microspheres had better adsorption capacity than conventional TiO2 powders, due to large surface area, large pore volume, and also a porous structure. The photocatalytic activity of TiO2 microspheres in aqueous suspension was evaluated using salicylic acid (SA) as a model substrate. It was found that the Langmuir,Hinshelwood model in its integral form described the kinetics of SA photocatalytic degradation in the TiO2 microsphere suspensions better than its simplified form as a first-order reaction model, since the significant substrate adsorption on the catalysts was not negligible. The kinetics of SA photocatalytic degradation with different initial concentrations and pH was further investigated. The experiments demonstrated that the change of pH could significantly affect the adsorption of SA in the TiO2 microsphere suspensions. The effects of substrate adsorption rate and photoreaction rate on the overall performance of photocatalytic degradation is also discussed on the basis of experimental data. Copyright © 2004 Society of Chemical Industry [source] Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coliMICROBIAL BIOTECHNOLOGY, Issue 5 2008Adam C. Fisher Summary Historically, the general secretory (Sec) pathway of Gram-negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin-arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N-termini upon reaching the periplasm and (iii) proteins fused to maltose-binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well-folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step. [source] A facile approach to surface graft vinyl acetate onto polyolefin articlesPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 9 2004Lin Tan Abstract A facile and environment friendly approach was developed to graft vinyl acetate (VAc) onto plastic articles in an aqueous solution using tert -butyl alcohol (TBA) as a compatiblizer and benzoyl peroxide (BPO) as an initiator. In a novel setup, excessive monomer suspended in a water phase, VAc could be conveniently grafted on the model substrate of low-density polyethylene (LDPE) film and the graft percentage (GP) could be developed up to 7.3%. Reaction temperature could increase GP significantly, while adding monomer over a critical volume did not influence GP. By adding some paradioxybenzene, i.e. 0.06,0.08% in VAc phase, homopolymer PVAc could be avoided practically, while graft polymerization proceeded favorably in aqueous solutions. It was proved by attenuated total reflection-infrared (ATR-IR) spectroscopy that grafted VAc was located mainly at the surface of the LDPE film and hydrophilic nature of both grafted and alcoholyzed films were improved via contact angle measurements. Copyright © 2004 John Wiley & Sons, Ltd. [source] The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S -adenosylmethionineTHE PLANT JOURNAL, Issue 5 2005Margret Sauter Summary Methylthioadenosine (MTA) is formed as a by-product of ethylene biosynthesis from S -adenosyl- l -methionine (AdoMet). The methionine cycle regenerates AdoMet from MTA. In two independent differential screens for submergence-induced genes and for 1-aminocyclopropane-1-carboxylic acid (ACC)-induced genes from deepwater rice (Oryza sativa L.) we identified an acireductone dioxygenase (ARD). OsARD1 is a metal-binding protein that belongs to the cupin superfamily. Acireductone dioxygenases are unique proteins that can acquire two different activities depending on the metal ion bound. Ectopically expressed apo-OsARD1 preferentially binds Fe2+ and reconstituted Fe-OsARD1 catalyzed the formation of 2-keto-pentanoate and formate from the model substrate 1,2-dihydroxy-3-ketopent-1-ene and dioxygen, indicating that OsARD1 is capable of catalyzing the penultimate step in the methionine cycle. Two highly homologous ARD genes were identified in rice. OsARD1 mRNA levels showed a rapid, early and transient increase upon submergence and after treatment with ethylene-releasing compounds. The second gene from rice, OsARD2, is constitutively expressed. Accumulation of OsARD1 transcript was observed in the same internodal tissues, i.e. the meristem and elongation zone, which were previously shown to synthesize ethylene. OsARD1 transcripts accumulated in the presence of cycloheximide, an inhibitor of protein synthesis, indicating that OsARD1 is a primary ethylene response gene. Promoter analysis suggests that immediate-early regulation of OsARD1 by ethylene may involve an EIN3-like transcription factor. OsARD1 is induced by low levels of ethylene. We propose that early feedback activation of the methionine cycle by low levels of ethylene ensures the high and continuous rates of ethylene synthesis required for long-term ethylene-mediated submergence adaptation without depleting the tissue of AdoMet. [source] Design of a cytochrome P450BM3 reaction system linked by two-step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenaseBIOTECHNOLOGY PROGRESS, Issue 5 2009Tsuyoshi Mouri Abstract A cytochrome P450BM3-catalyzed reaction system linked by a two-step cofactor regeneration was investigated in a cell-free system. The two-step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD+ -dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD+) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP+ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3-catalyzed reaction linked by the two-step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10-fold under initial reaction conditions. In contrast, a 10-fold increase in STH units resulted in about a 9-fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate-determining step. In the system lacking the two-step cofactor regeneration, 34% conversion of 50 ,M of a model substrate (p-nitrophenoxydecanoic acid) was attained using 50 ,M NADPH. In contrast, with the two-step cofactor regeneration, the same amount of substrate was completely converted using 5 ,M of oxidized cofactors (NAD+ and NADP+) within 1 h. Furthermore, a 10-fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD+ and NADP+. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Mechanistic Investigation of the Oxygen-Atom-Transfer Reactivity of Dioxo-molybdenum(VI) ComplexesCHEMISTRY - A EUROPEAN JOURNAL, Issue 28 2006Brian W. Kail Abstract The oxygen-atom-transfer (OAT) reactivity of [LiPrMoO2(OPh)] (1, LiPr=hydrotris(3-isopropylpyrazol-1-yl)borate) with the tertiary phosphines PEt3 and PPh2Me in acetonitrile was investigated. The first step, [LiPrMoO2(OPh)]+PR3,[LiPrMoO(OPh)(OPR3)], follows a second-order rate law with an associative transition state (PEt3, ,H,,=48.4 (±1.9) kJ,mol,1, ,S,,=,149.2 (±6.4) J,mol,1,K,1, ,G,,=92.9 kJ,mol,1; PPh2Me, ,H,,=73.4 (±3.7) kJ,mol,1, ,S,,=,71.9 (±2.3) J,mol,1,K,1, ,G,,=94.8 kJ,mol,1). With PMe3 as a model substrate, the geometry and the free energy of the transition state (TS) for the formation of the phosphine oxide-coordinated intermediate were calculated. The latter, 95 kJ,mol,1, is in good agreement with the experimental values. An unexpectedly large O-P-C angle calculated for the TS suggests that there is significant O-nucleophilic attack on the PC ,* in addition to the expected nucleophilic attack of the P on the MoO ,*. The second step of the reaction, that is, the exchange of the coordinated phosphine oxide with acetonitrile, [LiPrMoO(OPh)(OPR3)] + MeCN , [LiPrMoO(OPh)(MeCN)] + OPR3, follows a first-order rate law in MeCN. A dissociative interchange (Id) mechanism, with activation parameters of ,H,,=93.5 (±0.9) kJ,mol,1, ,S,,=18.2 (±3.3) J,mol,1,K,1, ,G,,=88.1 kJ,mol,1 and ,H,,=97.9 (±3.4) kJ,mol,1, ,S,,=47.3 (±11.8) J,mol,1,K,1, ,G,,=83.8 kJ,mol,1, for [LiPrMoO(OPh)(OPEt3)] (2,a) and [LiPrMoO(OPh)(OPPh2Me)] (2,b), respectively, is consistent with the experimental data. Although gas-phase calculations indicate that the MoOPMe3 bond is stronger than the MoNCMe bond, solvation provides the driving force for the release of the phosphine oxide and formation of [LiPrMoO(OPh)(MeCN)] (3). [source] Regulation of ,-Chymotrypsin Catalysis by Ferric Porphyrins and CyclodextrinsCHEMISTRY - AN ASIAN JOURNAL, Issue 4 2008Koji Kano Prof. Abstract Positively charged ,-chymotrypsin (ChT) formed a 1:1 complex with negatively charged 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) (FeTPPS) in phosphate buffer at pH,7.4 through electrostatic interaction. In spite of the large binding constant (K=4.8×105,M,1), FeTPPS could not completely inhibit the catalysis of ChT in the hydrolysis of the model substrate, N -succinyl- L -phenylalanine p -nitroanilide (SPNA). The degree of inhibition (60,%) was saturated at 1.6,equivalents of FeTPPS, which indicates that covering of the active site of ChT by FeTPPS was insufficient. The enzymatic activity lowered by FeTPPS was entirely recovered for the freshly prepared sample when the porphyrin on the protein surface was detached by per- O -methylated ,-cyclodextrin (TMe-,-CD), which formed a stable 1:2 inclusion complex with FeTPPS (K1=1.26×106,M,1, K2=6.3×104,M,1). FeTPPS gradually induced irreversible denaturation of ChT, and the denatured ChT further lost its catalytic ability. No repairing effect of TMe-,-CD was observed with irreversibly denatured ChT. A new reversible inhibitor, 5,10,15,20-tetrakis[4-(3,5-dicarboxyphenylmethoxy)phenyl]porphyrinato iron(III) (FeP8M), was then designed, and its inhibitory behavior was examined. FeP8M formed very stable 1:1 and 1:2 FeP8M/ChT complexes with ChT, the K1 and K2 values being 2.0×108 and 1.0×106,M,1, respectively. FeP8M effectively inhibited the ChT-catalyzed hydrolysis of SPNA (maximum degree of inhibition=85,%), and the activity of ChT was recovered by per- O -methylated ,-cyclodextrin. No irreversible denaturation of ChT occurred upon binding with FeP8M. The kinetic data support the observation that, for nonincubated samples, both inhibitors did not cause significant conformational change in ChT and inhibited the ChT activity by covering the active site of the enzyme. [source] Biotransformation of flobufen enantiomers in ruminant hepatocytes and subcellular fractionsCHIRALITY, Issue 10 2001Lenka Skálová Abstract Flobufen (F), a new antiinflammatory drug, has one chiral and one prochiral center in its structure. Reduction of rac - F, the principal biotransformation pathway, leads to the formation of four diastereoisomers of 4-dihydroflobufen (DHF). F was chosen as a model substrate for interspecies comparison of activity, stereospecificity, and stereoselectivity of biotransformation enzymes in fallow bucks, red deer stags, and roe bucks in vitro. Formation of F metabolites was examined in hepatocyte suspension and in subcellular fractions of liver homogenate. (+)-R -F, (,)-S -F and rac - F were used as substrates. After incubation of substrates, the amounts and ratios of DHF diastereoisomers and F enantiomers were assessed by HPLC, with (R,R)-ULMO and terguride-bonded columns. Considerable interspecies differences in stereoselectivity and stereospecificity of F reductases were found at the cellular and subcellular levels, although these ruminants are closely related. Chiral inversion of F enantiomers to their antipodes was detected in vitro in all ruminants tested, but individual species also differed in the direction and rate of this inversion. Chirality 13:760,764, 2001. © 2001 Wiley-Liss, Inc. [source] Elimination Mechanisms in the Aminolysis of Sulfamate Esters of the Type NH2SO2OC6H4X , Models of Enzyme InhibitorsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 24 2008William J. Spillane Abstract The kinetics of the reaction of 4-nitrophenyl sulfamate NH2SO2OC6H4NO2 -4 (1a) in acetonitrile (ACN) with a series of pyridines (pKa range ca. 8 units) and alicyclic amines (pKa range ca. 3.6 units) has been studied in the presence of excess amine at various temperatures. The compounds 1a,1f are important as model substrates for the medicinally important sulfamate esters 667-coumate and emate and analogues. Pseudo-first-order rate constants (kobsd.) have been obtained mainly by the release of 4-nitrophenol/4-nitrophenoxide. Slopes of plots of kobsd. vs. [amine] gave second-order rate constants (k2), and Brönsted plots were biphasic for the aminolysis (with alicyclic amines) with an initial slope ,1 = 0.53 and a subsequent slope ,2 = 0.19. The change in slope occurs near the first pKa of 1a (17.9) in ACN. Leaving-group effects were probed by using the same series of phenyl sulfamates, i.e. 1a,f and the alicyclic amines N -formylpiperazine and pyrrolidine. The reactions were considered to be dissociative in nature involving E2- and E1cB- type mechanisms with the phenyl sulfamate anion 2 being involved in pyridine and in the weaker alicyclic amines (,1 segment) and a phenyl sulfamate dianion 3 being involved with the stronger alicyclic bases (,2 segment). The calculation of Leffler indices (,) for bond-forming (base···H+) and bond-breaking (S,OAr) steps allows fuller interpretation of the mechanisms occurring, which are seen as having the N -sulfonylamines, HN=SO2 and ,N=SO2 on the reaction pathways leading to products. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] Regioflexibility in the Functionalization of Multiply Halogenated QuinolinesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 5 2004Marc Marull Abstract 4-Bromo-6-fluoro-2-(trifluoromethyl)quinoline (1) and 4-bromo-7-fluoro-2-(trifluoromethyl)quinoline (13) were selected as model substrates to explore under what conditions regiochemically exhaustive functionalization reactions can be carried out. This goal was achieved by using trimethylsilyl entities and iodine atoms as the sole auxiliary substituents. The organolithium intermediates could be generated and the protective groups removed without impairing the bromine atom present at the 4-position. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Effect of mutations in the ,5,,7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11)FEBS JOURNAL, Issue 21 2007Alexei S. Kasakov The human genome encodes ten different small heat shock proteins, each of which contains the so-called ,-crystallin domain consisting of 80,100 residues and located in the C-terminal part of the molecule. The ,-crystallin domain consists of six or seven ,-strands connected by different size loops and combined in two ,-sheets. Mutations in the loop connecting the ,5 and ,7 strands and conservative residues of ,7 in ,A-, ,B-crystallin and HSP27 correlate with the development of different congenital diseases. To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants. These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis-ANS bound to HSP22. Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis. Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking. The wild-type HSP22 possessed higher chaperone-like activity than their mutants when insulin or rhodanase were used as the model substrates. Because conservative Lys residues located in the ,5,,7 loop and in the ,7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases. [source] A Computational Study of the Sub-monolayer Growth of PentaceneADVANCED FUNCTIONAL MATERIALS, Issue 13 2006D. Choudhary Abstract A computational study of organic thin-film growth using a combination of ab,initio based energy calculations and kinetic Monte Carlo (KMC) simulations is provided. A lattice-based KMC model is used in which binding energies determine the relative rates of diffusion of the molecules. This KMC approach is used to present "landscapes" or "maps" that illustrate the possible structural outcomes of growing a thin film of small organic molecules, represented as a two-site dimer, on a substrate in which the strength of organic,substrate interactions is allowed to vary. KMC provides a mesoscopic-scale view of sub-monolayer deposition of organic thin films on model substrates, mapped out as a function of the flux of depositing molecules and the temperature of the substrate. The morphology of the crystalline thin films is shown to be a strong function of the molecule,molecule and molecule,substrate interactions. A rich variety of maps is shown to occur in which the small organic molecules either stand up or lie down in a variety of different patterns depending on the nature of the binding to the surface. In this way, it is possible to suggest how to tailor the substrate or the small organic molecule in order to create a desired growth habit. In order to demonstrate how this set of allowable maps is reduced in the case where the set of energy barriers between substrate and organic molecule are reliably known, we have used Gaussian,98 calculations to establish binding energies for the weak van der Waals interactions between a),pairs of pentacene molecules as a function of orientation and b),pentacene and two substrates, silicon surfaces passivated with cyclopentene molecules and a crystalline model of silicon dioxide. The critical nucleation size and the mode of diffusion of this idealized two-site dimer model for pentacene molecules are found to be in good agreement with experimental data. [source] The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Chirag B. Khatiwala Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre-osteoblastic MC3T3-E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen-activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3-E1 cells cultured on poly(ethylene glycol) (PEG)-based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance. J. Cell. Physiol. 211: 661,672, 2007. © 2007 Wiley-Liss, Inc. [source] Ligand effects upon deuterium exchange in arenes mediated by [Ir(PR3)2(cod)]+.BF4,JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 1 2004George J. Ellames Abstract A series of complexes of general form [Ir(PR3)2(cod)]+ has been prepared and used, without isolation, to mediate deuteration of a range of model substrates. The data suggest that, with many substrates, basicity of the phosphine ligands bound to iridium is an important factor influencing substrate selectivity and the efficiency of deuteration. In addition, the spectrum of activity of iridium complexes bearing pure donor ligands is different in many cases to that of complexes where the ligands are known to be ,-acids. Copyright © 2003 John Wiley & Sons, Ltd. [source] A new practical tritium labelling procedure using sodium borotritide and tetrakis(triphenylphosphine)palladium(0)JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 14 2001Tohru Nagasaki Abstract A simple, mild and versatile new tritium (3H) labelling method on a micro scale using sodium borotritide (NaB3H4) and a transition,metal complex catalyst is described. 3H-labelled compounds were prepared effectively by 3H hydrogenolysis of appendant functional groups in target compounds. The appendant functional group such as bromo, iodo or sulfonate in various target compounds can be replaced by tritium (3H) in moderate yields. The new method was established by optimization of the reaction conditions and examination of its applicability using four types of model substrates in tracer runs. Then, various drug candidates and ligands for drug discovery were labelled with tritium on a micro scale. The specific radioactivity of the 3H-labelled compounds used for the studies on receptor binding ranged from 12 to 20 Ci/mmol. Copyright © 2001 John Wiley & Sons, Ltd. [source] Relationship between esterase activity and acrinathrin and methiocarb resistance in field populations of western flower thrips, Frankliniella occidentalis,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 12 2006Ana C Maymó Abstract The western flower thrips, Frankliniella occidentalis (Pergande), is a serious pest in the south-east of Spain owing to its direct feeding on crops, transmission of the tomato spotted wilt virus and its very high level of resistance to insecticides. Mechanisms of resistance were examined using field populations of F. occidentalis with different susceptibilities to acrinathrin, methiocarb (selective insecticides), endosulfan, metamidophos and deltamethrin (broad-spectrum insecticides). Esterase activity towards ,-naphthyl acetate and p -nitrophenyl acetate in resistant strains was significantly higher than in the reference strain (MLFOM) for both model substrates. This higher activity was significantly correlated with acrinathrin and methiocarb resistance. Copyright © 2006 Society of Chemical Industry [source] Identification of degradation products formed during performic oxidation of peptides and proteins by high-performance liquid chromatography with matrix-assisted laser desorption/ionization and tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2005Jingquan Dai Oxidation of proteins with performic acid is extensively used to cleave disulfide bonds. Due to its efficiency and many other advantages it deserves more attention especially in proteomics as a method for sample treatment. However, some unwanted degradations can occur during performic oxidation. In this work the degradation products during performic oxidation of two peptides and bovine serum albumin as model substrates were explored by coupling high-performance liquid chromatography (HPLC) to matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOFMS). In addition to well-known modifications such as oxidation of tryptophan and oxidation and chlorination of tyrosine, novel degradation products including nonspecific cleavage after asparagine or tryptophan, formylation of lysine, and , -elimination of cysteine, were observed. Although almost all of these modification/degradation products except oxidation products of tryptophan were formed at sub-stoichiometric levels, they can cause confusion as a result of the sensitivity of mass spectrometry in analysis of the oxidized samples, especially in proteomics research. The results presented here will facilitate the interpretation of analytical data for performate-oxidized samples, and help to select appropriate methods for each unique sample. Copyright © 2005 John Wiley & Sons, Ltd. [source] Ring-Closing Olefin Metathesis on Ruthenium Carbene Complexes: Model DFT Study of StereochemistryCHEMISTRY - A EUROPEAN JOURNAL, Issue 13 2005Sergei F. Vyboishchikov Dr. Abstract Ring-closing metathesis (RCM) is the key step in a recently reported synthesis of salicylihalamide and related model compounds. Experimentally, the stereochemistry of the resulting cycloolefin (cis/trans) depends strongly on the substituents that are present in the diene substrate. To gain insight into the factors that govern the observed stereochemistry, density functional theory (DFT) calculations have been carried out for a simplified dichloro(2-propylidene)(imidazole-2-ylidene)ruthenium catalyst I, as well as for the real catalyst II with two mesityl substituents on the imidazole ring. Four model substrates are considered, which are closely related to the systems studied experimentally, and in each case, two pathways A and B are possible since the RCM reaction can be initiated by coordination of either of the two diene double bonds to the metal center. The first metathesis yields a carbene intermediate, which can then undergo a second metathesis by ring closure, metallacycle formation, and metallacycle cleavage to give the final cycloolefin complex. According to the DFT calculations, the stereochemistry is always determined in the second metathesis reaction, but the rate-determining step may be different for different catalysts, substrates, and pathways. The ancillary N-heterocyclic carbene ligand lies in the Ru-Cl-Cl plane in the simplified catalyst I, but is perpendicular to it in the real catalyst II, and this affects the relative energies of the relevant intermediates and transition states. Likewise, the introduction of methyl substituents in the diene substrates influences these relative energies appreciably. Good agreement with the experimentally observed stereochemistry is only found when using the real catalyst II and the largest model substrates in the DFT calculations. [source] Deposition of Barrier Layers for Thin Film Solar Cells Assisted by Bipolar Substrate BiasingPLASMA PROCESSES AND POLYMERS, Issue S1 2009Evelyn Häberle Abstract For the development of diffusion and insulation barriers for thin film solar cells on unpolished steel with a rough surface as substrate, investigations of the shape of deposited SiOx layers in dependence on an applied substrate biasing are carried out. Si-wafers with a well-defined surface structure in the range of micrometre are used as ,model' substrate. As a result, the deposition in the indentations of this surface is much higher in the case of a biased substrate. To determine the influence of the bias on the molecular structure, first investigations of the deposited layer without an applied bias are performed with in situ Fourier Transform InfraRed (FTIR) spectroscopy. Hence the molecular composition of the films is monitored during the deposition. In these spectra the Berreman effect occurs and is analysed. [source] | ||||||||||||||||||||||||||||