Discovery of potential cholesterol esterase inhibitors using in silico docking studies

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Thirumalaisamy Sivashanmugam

2013-08-01

Full Text Available New drug discovery is considered broadly in terms of two kinds of investiga-tional activities such as exploration and exploitation. This study deals with the evaluation of the cholesterol esterase inhibitory activity of flavonoids apigenin, biochanin, curcumin, diosmetin, epipervilline, glycitein, okanin, rhamnazin and tangeritin using in silico docking studies. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -7.08 kcal/mol to -5.64 kcal/mol when compared with that of the standard compound gallic acid (-4.11 kcal/mol. Intermolecular energy (-9.13 kcal/mol to -7.09 kcal/mol and inhibition constant (6.48 µM to 73.18 µM of the ligands also coincide with the binding energy. All the selected flavonoids contributed cholesterol esterase inhibitory activity, these molecular docking analyses could lead to the further develop-ment of potent cholesterol esterase inhibitors for the treatment of obesity.

Characterisation of a New Family of Carboxyl Esterases with an OsmC Domain.

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Mai-Britt V Jensen

Full Text Available Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries.

Identification of a Marine Bacillus Strain C5 and Parathion-Methyl Degradation Characteristics of the Extracellular Esterase B1

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Jianhua Hao

2014-01-01

Full Text Available A bacterial strain C5 that can produce new type of marine esterase was isolated and screened from marine sludge. According to 16S rRNA sequence analysis and physiological and biochemical experiments, the strain was identified as Bacillus subtilis. A single isozyme with a molecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE. On this basis, the mechanism of esterase B1 secreted by strain C5 degrading parathion-methyl was explored, and the effects of temperature and pH on the degradation rate were investigated. From the results, p-nitrophenol was one of the degradation products of B1 degrading parathion-methyl, and the best degradation effect could be achieved at the temperature of 40°C and the neutral pH value.

Exposure‐Response Model of Subcutaneous C1‐Inhibitor Concentrate to Estimate the Risk of Attacks in Patients With Hereditary Angioedema

Science.gov (United States)

Tortorici, Michael A.; Pawaskar, Dipti; Pragst, Ingo; Machnig, Thomas; Hutmacher, Matthew; Zuraw, Bruce; Cicardi, Marco; Craig, Timothy; Longhurst, Hilary; Sidhu, Jagdev

2018-01-01

Subcutaneous C1‐inhibitor (HAEGARDA, CSL Behring), is a US Food and Drug Administration (FDA)‐approved, highly concentrated formulation of a plasma‐derived C1‐esterase inhibitor (C1‐INH), which, in the phase III Clinical Studies for Optimal Management in Preventing Angioedema with Low‐Volume Subcutaneous C1‐inhibitor Replacement Therapy (COMPACT) trial, reduced the incidence of hereditary angioedema (HAE) attacks when given prophylactically. Data from the COMPACT trial were used to develop a repeated time‐to‐event model to characterize the timing and frequency of HAE attacks as a function of C1‐INH activity, and then develop an exposure–response model to assess the relationship between C1‐INH functional activity levels (C1‐INH(f)) and the risk of an attack. The C1‐INH(f) values of 33.1%, 40.3%, and 63.1% were predicted to correspond with 50%, 70%, and 90% reductions in the HAE attack risk, respectively, relative to no therapy. Based on trough C1‐INH(f) values for the 40 IU/kg (40.2%) and 60 IU/kg (48.0%) C1‐INH (SC) doses, the model predicted that 50% and 67% of the population, respectively, would see at least a 70% decrease in the risk of an attack. PMID:29316335

Esterase Isoenzyme Variants in Barley

DEFF Research Database (Denmark)

Hvid, S.; Nielsen, G.

1977-01-01

Gene symbols are proposed for 27 esterase isoenzyme alleles representing 10 loci in barley. Two new esterase loci, Est 9 and Est 10, each with an active and a silent allele, and three new alleles in previously described loci were found. A few chemical and physical characteristics of the different...... esterase isoenzyme systems were studied. The heat inactivation temperature differed for the isoenzymes coded by most of the loci, whereas the substrate and inhibitor specificity of the isoenzymes was less distinct. A possible relationship between some of the systems is discussed....

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes

OpenAIRE

1988-01-01

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular ...

Usefulness of C1 Esterase Inhibitor Protein Concentrate in the ...

African Journals Online (AJOL)

2018-04-04

Apr 4, 2018 ... 2018 Nigerian Journal of Clinical Practice | Published by Wolters Kluwer ‑ Medknow ... of this case report is to describe the lifesaving use of a novel C1‑INH protein ... edema of the upper lip, uvula, and tongue [Figure 1].

Functional classification of esterases from leaves of Aspidosperma polyneuron M. Arg. (Apocynaceae

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Carvalho Vanda Marilza de

2003-01-01

Full Text Available Polyacrylamide gel electrophoresis system (PAGE and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases fromAspidosperma polyneuron. The characterization of alpha- and beta-esterases from young leaves of A. polyneuron by the PAGE system showed fourteen esterase isozymes. The differential staining pattern showed that Est-2 isozyme hydrolyzes beta-naphthyl acetate; Est-6, Est-7 and Est-8 isozymes hydrolyze alpha-naphthyl acetate, and Est-1, Est-3, Est-4, Est-5, Est-9, Est-10, Est-11, Est-12, Est-13, and Est-14 isozymes hydrolyze both alpha- and b-naphthyl acetate. Inhibition pattern of a- and beta-esterases showed that Folidol is a more potent inhibitor that Malathion, while Thiamethoxan (an insecticide with organophosphorus-like action acts as an Est-4 and Est-6 inhibitor and induces the appearance of Est-5 and Est-7 isozymes as more intensely stained bands. Inhibition tests showed that OPC insecticides inhibit or activate plant esterases. Thus, plant esterases may be used as bioindicators to detect the presence and toxicity of residues of topically applied insecticides in agriculture and may be valuable for monitoring pollutants in the environment.

Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma

DEFF Research Database (Denmark)

Munkvad, S; Jespersen, J; Sidelmann, Johannes Jakobsen

1990-01-01

We present a new functional assay for the first complement component (C1) in plasma, based on its activation by inhibition of the C1-esterase inhibitor (C1-inh) when monospecific antiserum to C1-inh is added to the plasma. After maximal activation, we can determine the concentration of activated ...

HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene.

Science.gov (United States)

Kalmár, Lajos; Hegedüs, Tamás; Farkas, Henriette; Nagy, Melinda; Tordai, Attila

2005-01-01

Hereditary angioneurotic edema (HAE) is an autosomal dominant disorder characterized by episodic local subcutaneous and submucosal edema and is caused by the deficiency of the activated C1 esterase inhibitor protein (C1-INH or C1INH; approved gene symbol SERPING1). Published C1-INH mutations are represented in large universal databases (e.g., OMIM, HGMD), but these databases update their data rather infrequently, they are not interactive, and they do not allow searches according to different criteria. The HAEdb, a C1-INH gene mutation database (http://hae.biomembrane.hu) was created to contribute to the following expectations: 1) help the comprehensive collection of information on genetic alterations of the C1-INH gene; 2) create a database in which data can be searched and compared according to several flexible criteria; and 3) provide additional help in new mutation identification. The website uses MySQL, an open-source, multithreaded, relational database management system. The user-friendly graphical interface was written in the PHP web programming language. The website consists of two main parts, the freely browsable search function, and the password-protected data deposition function. Mutations of the C1-INH gene are divided in two parts: gross mutations involving DNA fragments >1 kb, and micro mutations encompassing all non-gross mutations. Several attributes (e.g., affected exon, molecular consequence, family history) are collected for each mutation in a standardized form. This database may facilitate future comprehensive analyses of C1-INH mutations and also provide regular help for molecular diagnostic testing of HAE patients in different centers.

Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

Science.gov (United States)

Rocchi, V; Janni, M; Bellincampi, D; Giardina, T; D'Ovidio, R

2012-03-01

Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Effect of halogenated benzenes on acetanilide esterase, acetanilide hydroxylase and procaine esterase in rats.

Science.gov (United States)

Carlson, G P; Dziezak, J D; Johnson, K M

1979-07-01

1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.

Esterase activity as a novel parameter of spore germination in Bacillus anthracis

International Nuclear Information System (INIS)

Ferencko, Linda; Cote, Mindy A.; Rotman, Boris

2004-01-01

Spores of Bacillus anthracis were shown to produce esterase activity about 4 min after exposure to conventional germinants such as combinations of amino acids and purine ribosides. Neither amino acids nor ribosides alone induce germination and esterase activity. Expression of esterase activity was chloramphenicol resistant, and correlated with loss of spore refractivity, a traditional parameter of early germination. Based on these observations, we hypothesized that esterase activity could be used as a novel parameter for quantifying early events during spore germination. To test this hypothesis, we measured expression of esterase activity under a variety of germinating conditions. Using diacetyl fluorescein as fluorogenic substrate of esterases, we demonstrated that esterase activity was invariably induced whenever spores were triggered by known germinants. Moreover, D-alanine, an inhibitor of L-alanine-mediated germination, was found to significantly inhibit expression of esterase activity. In terms of molecular mechanisms, esterase expression could represent activation of proteases at the onset of spore germination

Crystallization and preliminary X-ray analysis of a novel esterase Rv0045c from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Xu, Lipeng; Guo, Jiubiao; Zheng, Xiangdong; Wen, Tingyi; Sun, Fei; Liu, Siguo; Pang, Hai

2010-01-01

The novel esterase Rv0045c from M. tuberculosis was expressed and purified to homogeneity. The crystals of native and SeMet-labelled Rv0045c protein that were obtained diffracted to resolutions of 2.7 and 3.0 Å, respectively. The Rv0045c protein is predicted to be an esterase that is involved in lipid metabolism in Mycobacterium tuberculosis. The protein was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The Rv0045c protein crystals diffracted to a resolution of 2.7 Å using a synchrotron-radiation source and belonged to space group P3 1 or P3 2 , with unit-cell parameters a = b = 73.465, c = 48.064 Å, α = β = 90, γ = 120°. Purified SeMet-labelled Rv0045c protein was also crystallized and formed crystals that diffracted to a resolution of 3.0 Å using an in-house X-ray radiation source

Studies on the oxidizing system in Holt's medium for histochemical demonstration of esterase activity

DEFF Research Database (Denmark)

Kirkeby, S; Blecher, S R

1978-01-01

Esterase activity in guinea-pig thyroid and mouse epididymis epithelial cells has been studied using 5-bromoindoxyl acetate as substrate. The pattern of esterase activity in the thyroid of the guinea-pig is constant, irrespective of whether ferri-ferrocyanide (FFC) or certain copper compounds...... cells contain an esterase activity which is not inhibited by conventional SH blocking agents, nor by high concentrations of FFC. From these results it appears that the mode of action of FFC in Holt's medium is as follows. At low concentrations FFC appears to act primarily as a catalytic agent...... in oxidation of indoxyl to indigoid. At high concentration FFC acts as an inhibitor of guinea-pig thyroid esterase, by oxidation of SH groups in the active centre. The esterase of mouse epididymis cell type EH 1 is not subject to this inhibition by FFC, presumably because it does not contain accessible SH...

The search of the target of promotion: Phenylbenzoate esterase activities in hen peripheral nerve

International Nuclear Information System (INIS)

Moretto, A.; Nicolli, A.; Lotti, M.

2007-01-01

Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crude homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC 50 of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds

Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase.

Science.gov (United States)

Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

2013-12-01

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA. Copyright © 2013 Elsevier B.V. All rights reserved.

Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune.

Science.gov (United States)

Spániková, Silvia; Biely, Peter

2006-08-21

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.

A cold-induced pectin methyl-esterase inhibitor gene contributes negatively to freezing tolerance but positively to salt tolerance in Arabidopsis.

Science.gov (United States)

Chen, Jian; Chen, Xuehui; Zhang, Qingfeng; Zhang, Yidan; Ou, Xiangli; An, Lizhe; Feng, Huyuan; Zhao, Zhiguang

2018-03-01

Plant pectin methyl-esterase (PME) and PME inhibitor (PMEI) belong to large gene families whose members are proposed to be widely involved in growth, development, and stress responses; however, the biological functions of most PMEs and PMEIs have not been characterized. In this study, we studied the roles of CbPMEI1, a cold-induced pectin methyl-esterase inhibitor (PMEI) gene from Chorispora bungeana, under freezing and salt stress. The putative CbPMEI1 peptide shares highest similarity (83%) with AT5G62360 (PMEI13) of Arabidopsis. Overexpression of either CbPMEI1 or PMEI13 in Arabidopsis decreased tissue PME activity and enhanced the degree of methoxylation of cell wall pectins, indicating that both genes encode functional PMEIs. CbPMEI1 and PMEI13 were induced by cold but repressed by salt stress and abscisic acid, suggesting distinct roles of the genes in freezing and salt stress tolerance. Interestingly, transgenic Arabidopsis plants overexpressing CbPMEI1 or PMEI13 showed decreased freezing tolerance, as indicated by survival and electrolyte leakage assays. On the other hand, the salt tolerance of transgenic plants was increased, showing higher rates of germination, root growth, and survival under salinity conditions as compared with non-transgenic wild-type plants. Although the transgenic plants were freezing-sensitive, they showed longer roots than wild-type plants under cold conditions, suggesting a role of PMEs in balancing the trade-off between freezing tolerance and growth. Thus, our study indicates that CbPMEI1 and PMEI13 are involved in root growth regulation under cold and salt stresses, and suggests that PMEIs may be potential targets for genetic engineering aimed to improve fitness of plants under stress conditions. Copyright © 2018 Elsevier GmbH. All rights reserved.

The activity of non-specific esterase in the thyroid epithelial cells of the guinea pig as influenced by various inhibitors and activators. A histochemical study

DEFF Research Database (Denmark)

Kirkeby, S

1976-01-01

The action of various inhibitors and activators upon esterase activity in the thyroid epithelial cells is demonstrated. The agents used were triorthocresylphosphate (TOCP), parachloromercuribenzoate (PCMB), Arsanillic acid, p-nitrophenyl dimethyl carbamate and bis p-nitrophenyl phosphate. TOCP wa...

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

Science.gov (United States)

Rejón, Juan D; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J

2012-10-01

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

Esterase profile of human masseter muscle

DEFF Research Database (Denmark)

Kirkeby, S; Moe, D; Vilmann, H

1988-01-01

The esterase profile of fresh human masseter muscle was investigated by use of histochemistry and electrophoresis. The histochemical methods included reactions for alpha-naphthyl esterase, myofibrillar ATPase, reverse myofibrillar ATPase and succinic dehydrogenase. In frozen sections of the muscle...... the coloured reaction product for esterases was present both as a diffuse sarcoplasmic coloration and as distinct granules. The intensity of diffuse reaction was used to classify the muscle fibres as strongly, moderately and weakly reacting. The fibres with strong esterase activity belonged to Type I and ii......C. iM and Type II A fibres showed a moderate esterase reaction and Type II B fibres had a low activity. The electrophoretic gels stained for esterase activity showed that the human masseter muscle possesses a slow migrating double band with high enzyme activity and a cascade of faster migrating...

In vitro comparison of rat and chicken brain neurotoxic esterase

International Nuclear Information System (INIS)

Novak, R.; Padilla, S.

1986-01-01

A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chicken brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterases showed that rat esterases were more sensitive than chicken to paraoxon inhibition at concentrations less than or equal to microM and superimposable with chicken esterases at concentrations of 2.5-1000 microM. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon concentration of 100 microM (mipafox concentration: 0-1000 microM) resulted in a mipafox I50 of 7.3 microM for chicken brain NTE and 11.6 microM for rat brain NTE. NTE (i.e., paraoxon-resistant, mipafox-sensitive esterase activity) comprised 80% of chicken and 60% of rat brain paraoxon-resistant activity with the specific activity of chicken brain NTE approximately twice that of rat brain NTE. The pH maxima for NTE from both species was similar showing broad, slightly alkaline optima from pH 7.9 to 8.6. [ 3 H]Diisopropyl phosphorofluoridate (DFP)-labeled NTE from the brains of both species had an apparent mol wt of 160,000 measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In conclusion, NTE from both species was very similar, with the mipafox I50 for rat NTE within the range of reported values for chicken and human NTE, and the inhibitor parameters of the chicken NTE assay were applicable for the rat NTE assay

Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica)

OpenAIRE

Kanneganti, Vydehi; Gupta, Aditya Kumar

2009-01-01

Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza...

Inhibition of pectin methyl esterase activity by green tea catechins.

Science.gov (United States)

Lewis, Kristin C; Selzer, Tzvia; Shahar, Chen; Udi, Yael; Tworowski, Dmitry; Sagi, Irit

2008-10-01

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.

The effect of EDTA and metal cations on the 5-bromoindoxyl acetate esterase activity in the thyroid of the guinea pig

DEFF Research Database (Denmark)

Kirkeby, S

1976-01-01

Miscellaneous metal cations and EDTA have been used as activators and inhibitors of esterase activity in the thyroid of the guinea-pig. The results indicate that the 5-bromoiondoxyl acetate esterase in the epithelial cells probably consists of two different A-esterase isoenzymes, one present...

Inhibition of Pectin Methyl Esterase Activity By Green Tea Catechins

OpenAIRE

Sagi, Irit; Lewis, Kristin; Tworowski, Dmitry; Shahar, Chen; Selzer, Tzvia

2008-01-01

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin ...

Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165

Directory of Open Access Journals (Sweden)

Deepthy Alex

2014-01-01

Full Text Available Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a Km and Vmax of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol.

Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica).

Science.gov (United States)

Kanneganti, Vydehi; Gupta, Aditya Kumar

2009-04-01

Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza sativaPectin Methyl Esterase 1). It contained the structural arrangement GXYXE and GXXDFIF, found in the active groups of all PMEs. OsPME1 gene product shared varying identities, ranging from 52 % to 33 % with PMEs from other plant species belonging to Brassicaceae, Fabaceae, Amaranthaceae and Funariaceae. Southern blot analysis indicated that PME1 exists as a single copy in the rice genome. Expression pattern analysis revealed that OsPME1 is expressed only in pollen grains, during the later stages of their development and was also regulated by various abiotic stress treatments and phytohormones. Functional characterization of this pollen specific PME from rice would enable us to understand its role in pollen development.

Esterase variation in Turkish white-toothed shrews (Crocidura: Record of a trimeric esterase

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Tez C.

2009-01-01

Full Text Available This study focuses on esterase variation of the genus Crocidura in Turkey. A total of 248 white-toothed shrews were analyzed by means of cellulose acetate gel electrophoresis. Liver tissue and alfa naphthyl acetate were used to investigate esterase variation in Turkish white-toothed shrews. A different esterase banding pattern was found in one Crocidura individual. This phenotype had four anodally migrated bands on cellulose acetate gel. The Crocidura individual displaying the given phenotype was identified as Crocidura suaveolens. The different esterase banding pattern observed in this study is considered to be a result of the trimeric structure of esterase in the lesser white-toothed shrew (Crocidura suaveolens.

Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC.

Science.gov (United States)

Dilokpimol, Adiphol; Mäkelä, Miia R; Mansouri, Sadegh; Belova, Olga; Waterstraat, Martin; Bunzel, Mirko; de Vries, Ronald P; Hildén, Kristiina S

2017-07-25

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

NARCIS (Netherlands)

Levisson, M.; Han, G.W.; Deller, M.C.; Hendriks, S.N.A.; Oost, van der J.; Kengen, S.W.M.

2012-01-01

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity

Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. IV: Cellular localization of androgen sensitive nonspecific esterase in the epididymis

DEFF Research Database (Denmark)

Kirkeby, S; Blecher, S R

1981-01-01

Nonspecific esterase of mouse epididymis has previously been studied histochemically, using alpha naphthyl-acetate and 5-bromoindoxyl acetate techniques, as well as certain inhibitors. Epithelial cell types of the epididymis have been characterized, and certain esterase isozymes in a particular...

A Novel Cold Active Esterase from a Deep Sea Sponge Stelletta normani Metagenomic Library

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Erik Borchert

2017-09-01

Full Text Available Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. Due to the widespread applications of lipolytic enzymes in various industrial applications, there continues to be an interest in novel esterases with unique properties. Marine ecosystems have long been acknowledged as a significant reservoir of microbial biodiversity and in particular of bacterial enzymes with desirable characteristics for industrial use, such as for example cold adaptation and activity in the alkaline pH range. We employed a functional metagenomic approach to exploit the enzymatic potential of one particular marine ecosystem, namely the microbiome of the deep sea sponge Stelletta normani. Screening of a metagenomics library from this sponge resulted in the identification of a number of lipolytic active clones. One of these encoded a highly, cold-active esterase 7N9, and the recombinant esterase was subsequently heterologously expressed in Escherichia coli. The esterase was classified as a type IV lipolytic enzyme, belonging to the GDSAG subfamily of hormone sensitive lipases. Furthermore, the recombinant 7N9 esterase was biochemically characterized and was found to be most active at alkaline pH (8.0 and displays salt tolerance over a wide range of concentrations. In silico docking studies confirmed the enzyme's activity toward short-chain fatty acids while also highlighting the specificity toward certain inhibitors. Furthermore, structural differences to a closely related mesophilic E40 esterase isolated from a marine sediment metagenomics library are discussed.

Overview of hereditary angioedema caused by C1-inhibitor deficiency: assessment and clinical management.

Science.gov (United States)

Bork, K; Davis-Lorton, M

2013-02-01

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1-INH) is a rare, autosomal-dominant disease. HAE-C1-INH is characterized by recurrent attacks of marked, diffuse, nonpitting and nonpruritic skin swellings, painful abdominal attacks, and laryngeal edema. The extremities and the gastrointestinal tract are most commonly affected. Swelling of the upper respiratory mucosa poses the greatest risk because death from asphyxiation can result from laryngealedema. HAE-C1-INH attacks are variable, unpredictable, and may be induced by a variety of stimuli, including stress or physical trauma. Because the clinical presentation of HAE-C1-INH is similar to other types of angioedema, the condition may be a challenge to diagnose. Accurate identification of HAE-C1-INH is critical in order to avoid asphyxiation by laryngeal edema and to improve the burden of disease. Based on an understanding of the underlying pathophysiology of IHAE-C1-INH, drugs targeted specifically to the disease, such as C1-inhibitor therapy, bradykinin B2-receptor antagonists, and kallikrein-inhibitors, have become available for both treatment and prevention of angioedema attacks. This article reviews the clinical features, differential diagnosis, and current approaches to management of HAE-C1-INH.

Synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid vinyl-ACCA) derivatives: key intermediates for the preparation of inhibitors of the hepatitis C virus NS3 protease.

Science.gov (United States)

Beaulieu, Pierre L; Gillard, James; Bailey, Murray D; Boucher, Colette; Duceppe, Jean-Simon; Simoneau, Bruno; Wang, Xiao-Jun; Zhang, Li; Grozinger, Karl; Houpis, Ioannis; Farina, Vittorio; Heimroth, Heidi; Krueger, Thomas; Schnaubelt, Jürgen

2005-07-22

(1R,2S)-1-Amino-2-vinylcyclopropanecarboxylic acid (vinyl-ACCA) is a key building block in the synthesis of potent inhibitors of the hepatitis C virus NS3 protease such as BILN 2061, which was recently shown to dramatically reduce viral load after administration to patients infected with HCV genotype 1. We have developed a scalable process that delivers derivatives of this unusual amino acid in >99% ee. The strategy was based on the dialkylation of a glycine Schiff base using trans-1,4-dibromo-2-butene as an electrophile to produce racemic vinyl-ACCA, which was subsequently resolved using a readily available, inexpensive esterase enzyme (Alcalase 2.4L). Factors that affect diastereoselection in the initial dialkylation steps were examined and the conditions optimized to deliver the desired diastereomer selectively. Product inhibition, which was encountered during the enzymatic resolution step, initially resulted in prolonged cycle times. Enrichment of racemic vinyl-ACCA through a chemical resolution via diastereomeric salt formation or the use of forcing conditions in the enzymatic reaction both led to improvements in throughput and the development of a viable process. The chemistry described herein was scaled up to produce multikilogram quantities of this building block.

Three feruloyl esterases in Cellulosilyticum ruminicola H1 act synergistically to hydrolyze esterified polysaccharides.

Science.gov (United States)

Li, Jiabao; Cai, Shichun; Luo, Yuanming; Dong, Xiuzhu

2011-09-01

Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.

Cholesterol esterase activity of human intestinal mucosa

International Nuclear Information System (INIS)

Ponz de Leon, M.; Carubbi, F.; Di Donato, P.; Carulli, N.

1985-01-01

It has been suggested that cholesterol absorption in humans is dependent on bile acid pool composition and that expansion of the cholic acid pool size is followed by an increase of the absorption values. Similar observations were reported in rats. In the present study, therefore, the authors investigated some general properties of human intestinal cholesterol esterase, with particular emphasis on the effect of bile acids on this enzymatic activity. Twenty-nine segments of small intestine were taken during operations; the enzymatic activity was studied by using mucosal homogenate as a source of enzyme and oleic acid, cholesterol, and 14 C-labeled cholesterol as substrates. The time-activity relationship was linear within the first two hours; optimal pH for esterification ranged between 5 and 6.2. There was little difference between the esterifying activity of the jejunal and ileal mucosa. Esterification of cholesterol was observed with all the investigated fatty acids but was maximal with oleic acid. Bile acids did not affect cholesterol esterase activity when present in the incubation mixture at 0.1 and 1.0 mM; the enzymatic activity, however, was significantly inhibited when bile acids were added at 20 mM. In conclusion, this study has shown that the human intestinal mucosa possesses a cholesterol esterase activity; at variance with the rat, however, the human enzyme does not seem to be stimulated by trihydroxy bile acids

Pectin methyl esterase activity in apple and orange pulps

International Nuclear Information System (INIS)

Abdullaev, A.; Djumaev, B.B.; Djumaev, N.B.; Mukhidinov, Z.K.

2008-01-01

The results of pectin methyl esterase activity from apple, orange pulp and orange peel depending of ph and temperature are discussed. It's shown that the methyl esterase activity form apple and orange pulps higher in range of temperatures from +37...+60 d ig C . The analysis of dependence of its activity from ph has shown that in both case the enzyme activity increase with increase of ph

Preliminary X-ray analysis of twinned crystals of the Q88Y25-Lacpl esterase from Lactobacillus plantarum WCFS1

International Nuclear Information System (INIS)

Álvarez, Yanaisis; Esteban-Torres, María; Acebrón, Iván; Rivas, Blanca de las; Muñoz, Rosario; Martínez-Ripoll, Martín; Mancheño, José M.

2011-01-01

The Q88Y25-Lacpl esterase from L. plantarum WCFS1 has been recombinantly expressed, purified and crystallized. A native diffraction data set has been collected to 2.24 Å resolution. Q88Y25-Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His 6 -tagged Q88Y25-Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His 6 -tagged Q88Y25-Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å

Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

Science.gov (United States)

Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

2013-01-01

A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

Hydrolysis of Wheat Arabinoxylan by Two Acetyl Xylan Esterases from Chaetomium thermophilum

DEFF Research Database (Denmark)

Tong, Xiaoxue; Lange, Lene; Grell, Morten Nedergaard

2015-01-01

The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has...

Esterase reactions in acute myelomonocytic leukemia.

Science.gov (United States)

Kass, L

1977-05-01

Specific and nonspecific esterase reactions of bone marrow cells from 14 patients with untreated acute myelomonocytic leukemia and six patients with acute histiomonocytic leukemia were examined. The technic for esterase determination permitted simultaneous visualization of both esterases on the same glass coverslip containing the marrow cells. In cases of acute histiomonocytic leukemia, monocytes, monocytoid hemohistioblasts and undifferentiated blasts stained intensely positive for nonspecific esterase, using alpha-naphthyl acetate as the substrate. No evidence of specific esterase activity using naphthol ASD-chloroacetate as the substrate and fast blue BBN as the dye coupler was apparent in these cells. In all of the cases of acute myelomonocytic leukemia, both specific and nonspecific esterases were visualized within monocytes, monocytoid cells, and granulocytic cells that had monocytoid-type nuclei. Nonspecific esterase activity was not observed in polymorphonuclear leukocytes in cases of myelomonocytic leukemia. The results support a current viewpoint that acute myelomonocytic leukemia may be a variant of acute myeloblastic leukemia, and that cytochemically, many of the leukemic cells in myelomonocytic leukemia share properties of both granulocytes and monocytes.

Studies on esterase isozymes and mycelium growth speed of ganoderma lucidum carried by Shenzhou spaceship

International Nuclear Information System (INIS)

Qi Jianjun; Chen Xiangdong; Lan Jin

2002-01-01

The esterase isozymes and mycelium growth speed of four Ganoderma lucidum strains carried by Shenzhou spaceship were studied. The results showed that different effects occurred to esterase and mycelium growth speed. The SX, S3 esterase band had changed compared with their control CX, C3, respectively, but there were no differences between SH and CH, S4 and C4. The growth speed of S4 strain was faster than its control C4, SX strain lower than its control CX, and there were no difference between SH and CH, S3 and C3

Larvicides and acetylcholinesterase inhibitors from Kalanchoe species

International Nuclear Information System (INIS)

Trevisan, Maria Teresa Salles; Bezerra, Maria Zeneide Barbosa; Santiago, Gilvandete Maria Pinheiro; Feitosa, Chistiane Mendes; Verpoorte, Robert; Gorlaeus Laboratories, Leiden; Braz Filho, Raimundo

2006-01-01

Acetylcholine esterase inhibitors are successfully used to treat the symptoms of Alzheimer's disease. Extracts of three Kalanchoe species (K. brasiliensis, K. pinnata and K. gastonis-bornieri) showed acetylcholine esterase inhibitory effects and a toxic effect on Aedes aegypti larvae. Here we describe the bioassay guided fractionation of extracts of the most active extracts (K. brasiliensis) which resulted in the isolation of an active mixture of three flavonoids: 8-methoxyquercetin, 3,7-di-O-rhamnopyranoside and 8-methoxykaempferol-3,7-di-O-rhamnopyranoside. On TLC these flavonoids showed an acetylcholine esterase inhibitory effect. (author)

A p-coumaroyl esterase from Rhizoctonia solani with a pronounced chlorogenic acid esterase activity.

Science.gov (United States)

Nieter, Annabel; Kelle, Sebastian; Linke, Diana; Berger, Ralf G

2017-07-25

Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris. Recombinant enzyme production on the 5-L bioreactor scale (Rs pCAE: 3245UL -1 ) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs pCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity. The Rs pCAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs pCAE together with the lack of sequence similarity to known FAEs or pCAEs suggested that the Rs pCAE represents a new type of enzyme. Hydroxycinnamic acids were released from agro-industrial side-streams, such as destarched wheat bran (DSWB), sugar beet pectin (SBP) and coffee pulp (CP). Overnight incubation of coffee pulp with the Rs pCAE resulted in the efficient release of p-coumaric (100%), caffeic (100%) and ferulic acid (85%) indicating possible applications for the valorization of food processing wastes and for the enhanced degradation of lignified biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Study of Esterase and Hemolytic Activities in Clinically Important Candida Species, Isolated From Oral Cavity of Diabetic and Non-diabetic Individuals.

Science.gov (United States)

Fatahinia, Mahnaz; Poormohamadi, Farzad; Zarei Mahmoudabadi, Ali

2015-03-01

Diabetes mellitus as a chronic metabolic disease occurs in patients with partial or complete deficiency of insulin secretion or disorder in action of insulin on tissue. The disease is known to provide conditions for overgrowth of Candida species. Candida spp. cause candidiasis by many virulence factors such as esterase, hemolysin and phospholipase. This study aimed to compare esterase and hemolytic activity in various Candida species isolated from oral cavity of diabetic and non-diabetic individuals. Swab samples were taken from 95 patients with diabetes (35 men and 60 women) and 95 normal persons (42 men and 53 women) and cultured on Sabouraud dextrose agar. Identification of isolated yeasts was performed by germ tube test, morphology on CHROMagar Candida medium, corn meal agar and ability to grow at 45°C. Hemolysin activity was evaluated using blood plate assay and esterase activity was determined using the Tween 80 opacity test. Different Candida species were isolated from 57 (60%) diabetic and 24 (25%) non-diabetic individuals. Esterase activity was detected in all Candida isolates. Only 21.6% of C. albicans from patients with diabetes had esterase activity as + 3, while it ranged from + 1 to + 2 in others. Hemolytic activity was determined in C. albicans, C. dubliniensis, C. glabrata and C. krusei as 0.79, 0.58, 0.66 and 0.74, respectively. Hemolytic activity was significantly different in the two groups of diabetics and non-diabetics. Oral carriage of C. albicans in the diabetic group (n = 42; 66.7%) was significantly greater than the control group (n = 16; 57.1%). Esterase activity of C. albicans in diabetic group was higher than non-diabetic group. Although C. albicans remains the most frequently pathogenic yeast for human, but other species are increasing.

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

OpenAIRE

Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-01-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the ...

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

Directory of Open Access Journals (Sweden)

Leda Maria Fortes Gottschalk

2013-01-01

Full Text Available The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 ºC and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L, which was not detected in the T. reesei culture.

Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

International Nuclear Information System (INIS)

Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu

2007-01-01

Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-κB activation and nuclear translocation in an IκBα-dependent manner. The inhibitory effects were associated with reduction of inhibitor IκB kinase activity and stabilization of the NF-κB inhibitor IκB. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations

Crystallization and preliminary X-ray diffraction analysis of ybfF, a new esterase from Escherichia coli K12

Energy Technology Data Exchange (ETDEWEB)

Park, Suk-Youl; Lee, Sang-Hak; Lee, Jieun; Jung, Che-Hun; Kim, Jeong-Sun, E-mail: jsunkim@chonnam.ac.kr [Department of Chemistry and Institute of Basic Sciences, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

2007-12-01

The crystallization of ybfF, a new esterase from E. coli, and the collection of diffraction data to 1.1 Å resolution are reported. The product of the recently discovered ybfF gene, which belongs to the esterase family, does not show high sequence similarity to other esterases. To provide the molecular background to the enzymatic mechanism of the ybfF esterase, the ybfF protein from Escherichia coli K12 (Ec-ybfF) was cloned, expressed and purified. The Ec-ybfF protein was crystallized from 60% Tacsimate and 0.1 M bis-Tris propane buffer pH 7.0. Diffraction data were collected to 1.10 Å resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.09, b = 90.71, c = 92.88 Å. With two Ec-ybfF molecules in the asymmetric unit, the crystal volume per unit protein weight is 2.17 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 42%.

Gender differences in the activities of aspirin-esterases in rat tissues

Directory of Open Access Journals (Sweden)

Benedito M.A.C.

1998-01-01

Full Text Available The activities of aspirin (acetylsalicylic acid-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5 and II (assayed at pH 7.4 activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 ± 4.8 (N = 8 and females 29.3 ± 4.2 (N = 8 nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 ± 4.1 (N = 8 and females 26.1 ± 4.5 (N = 8 nmol of salicylic acid formed min-1 mg protein-1, P<0.001. In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 ± 0.06 (N = 6 and females 1.18 ± 0.11 (N = 6 nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 1.03 ± 0.13 (N = 6 and females 1.34 ± 0.11 (N = 6 nmol of salicylic acid formed min-1 mg protein-1, P<0.001. In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.

Chaperone-like activities of α-synuclein: α-Synuclein assists enzyme activities of esterases

International Nuclear Information System (INIS)

Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo; Doohun Kim, T.

2006-01-01

α-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of α-synuclein has not yet been known. Here we have shown that α-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of α-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with α-synuclein. Our results indicate that α-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo

Genome-wide analysis of esterase-like genes in the striped rice stem borer, Chilo suppressalis.

Science.gov (United States)

Wang, Baoju; Wang, Ying; Zhang, Yang; Han, Ping; Li, Fei; Han, Zhaojun

2015-06-01

The striped rice stem borer, Chilo suppressalis, a destructive pest of rice, has developed high levels of resistance to certain insecticides. Esterases are reported to be involved in insecticide resistance in several insects. Therefore, this study systematically analyzed esterase-like genes in C. suppressalis. Fifty-one esterase-like genes were identified in the draft genomic sequences of the species, and 20 cDNA sequences were derived which encoded full- or nearly full-length proteins. The putative esterase proteins derived from these full-length genes are overall highly diversified. However, key residues that are functionally important including the serine residue in the active site are conserved in 18 out of the 20 proteins. Phylogenetic analysis revealed that most of these genes have homologues in other lepidoptera insects. Genes CsuEst6, CsuEst10, CsuEst11, and CsuEst51 were induced by the insecticide triazophos, and genes CsuEst9, CsuEst11, CsuEst14, and CsuEst51 were induced by the insecticide chlorantraniliprole. Our results provide a foundation for future studies of insecticide resistance in C. suppressalis and for comparative research with esterase genes from other insect species.

A new microplate screening method for the simultaneous activity quantification of feruloyl esterases, tannases, and chlorogenate esterases.

Science.gov (United States)

Ramírez, L; Arrizon, J; Sandoval, G; Cardador, A; Bello-Mendoza, R; Lappe, P; Mateos-Díaz, J C

2008-12-01

Feruloyl, chlorogenate esterases, and tannases are enzymes useful in phenolic modifications of pharmaceutical relevance as protectors against several degenerative human diseases. Therefore, there is a growing interest in discovering new sources of these enzymes. However, traditional methods for their activity measurements are time-consuming and poorly adapted for high-throughput screening. In this study, a successful new microplate high-throughput screening method for the simultaneous quantification of all mentioned activities is demonstrated. This method allows the detection of activities as low as 1.7 mU ml(-1). Furthermore, reaction rates increased proportionally with the amount of enzyme added, and no interferences with the other commercial hydrolases tested were found. The utility of the method was demonstrated after simultaneously screening feruloyl, chlorogenate esterase, and tannase activities in solid state fermentation extracts obtained during the kinetics of production of 20 fungal strains. Among these, seven strains were positive for at least one of the esterase activities tested. This result shows the potential for the rapid routine screening assays for multiple samples of moderate low to high enzymatic levels.

Cytological localization of adenosine kinase, nucleoside phosphorylase-1, and esterase-10 genes on mouse chromosome 14

International Nuclear Information System (INIS)

Samuelson, L.C.; Farber, R.A.

1985-01-01

The authors have determined the regional locations on mouse chromosome 14 of the genes for mouse adenosine kinase (ADK), nucleoside phosphorylase- 1 (NP-1), and esterase-10 (ES-10) by analysis of rearranged mouse chromosomes in gamma-irradiated Chinese hamster X mouse hybrid cell lines. Irradiated clones were screened for expression of the murine forms of these enzymes; segregant clones that expressed only one or two of the three markers were karyotyped. The patterns of enzyme expression in these segregants were correlated with the presence of rearranged chromosomes. The Adk gene was localized to bands A2 to B, Np-1 to bands B to C1, and Es-10 to bands D2 to E2

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Science.gov (United States)

Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

2011-09-01

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

International consensus on the diagnosis and management of pediatric patients with hereditary angioedema with C1 inhibitor deficiency

OpenAIRE

Farkas, H.; Martinez?Saguer, I.; Bork, K.; Bowen, T.; Craig, T.; Frank, M.; Germenis, A. E.; Grumach, A. S.; Luczay, A.; Varga, L.; Zanichelli, A.; Aberer, Werner; Andrejevic, Sladjana; Aygoeren?P?rs?n, Emel; Banerji, Alena

2016-01-01

BACKGROUND: The consensus documents published to date on hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) have focused on adult patients. Many of the previous recommendations have not been adapted to pediatric patients. We intended to produce consensus recommendations for the diagnosis and management of pediatric patients with C1-INH-HAE.METHODS: During an expert panel meeting that took place during the 9th C1 Inhibitor Deficiency Workshop in Budapest, 2015 (www.haenet.hu), ped...

Discovery of potent 1H-imidazo[4,5-b]pyridine-based c-Met kinase inhibitors via mechanism-directed structural optimization.

Science.gov (United States)

An, Xiao-De; Liu, Hongyan; Xu, Zhong-Liang; Jin, Yi; Peng, Xia; Yao, Ying-Ming; Geng, Meiyu; Long, Ya-Qiu

2015-02-01

Starting from our previously identified novel c-Met kinase inhibitors bearing 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one scaffold, a global structural exploration was conducted to furnish an optimal binding motif for further development, directed by the enzyme inhibitory mechanism. First round SAR study picked two imidazonaphthyridinone frameworks with 1,8- and 3,5-disubstitution pattern as class I and class II c-Met kinase inhibitors, respectively. Further structural optimization on type II inhibitors by truncation of the imidazonaphthyridinone core and incorporation of an N-phenyl cyclopropane-1,1-dicarboxamide pharmacophore led to the discovery of novel imidazopyridine-based c-Met kinase inhibitors, displaying nanomolar enzyme inhibitory activity and improved Met kinase selectivity. More significantly, the new chemotype c-Met kinase inhibitors effectively inhibited Met phosphorylation and its downstream signaling as well as the proliferation of Met-dependent EBC-1 human lung cancer cells at submicromolar concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

A cold active (2R,3R)-(-)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa.

Science.gov (United States)

Zimmer, Christian; Platz, Tanja; Cadez, Neza; Giffhorn, Friedrich; Kohring, Gert-Wieland

2006-11-01

In a screening procedure a pink-colored yeast was isolated from enrichment cultures with (2R,3R)-(-)-di-O-benzoyl-tartrate (benzoyl-tartrate) as the sole carbon source. The organism saar1 was identified by morphological, physiological, and 18S ribosomal DNA/internal transcribed spacer analysis as Rhodotorula mucilaginosa, a basidiomycetous yeast. During growth the yeast hydrolyzed the dibenzoyl ester stoichiometrically to the monoester using the separated benzoate as the growth substrate, before the monoester was further cleaved into benzoate and tartrate, which were both metabolized. The corresponding benzoyl esterase was purified from the culture supernatant and characterized as a monomeric glycosylated 86-kDa protein with an optimum pH of 7.5 and an optimum temperature of 45 degrees C. At 0 degrees C the esterase still exhibited 20% of the corresponding activity at 30 degrees C, which correlates it to psychrophilic enzymes. The esterase could hydrolyze short chain p-nitrophenyl-alkyl esters and several benzoyl esters like benzoyl-methyl ester, ethylene-glycol-dibenzoyl ester, phenyl-benzoyl ester, cocaine, and 1,5-anhydro-D: -fructose-tribenzoyl ester. However feruloyl-ethyl ester was not hydrolyzed. The activity characteristics let the enzyme appear as a promising tool for synthesis of benzoylated compounds for pharmaceutical, cosmetic, or fine chemical applications, even at low temperatures.

A case of tongue edema associated with radiation-induced ulcer with low level of C1 inhibitor activity

International Nuclear Information System (INIS)

Hata, Tsuyoshi; Hosoda, Masaru

2003-01-01

A 66-year-old man became aware of sudden swelling of the tongue with swallowing disturbance. He had a medical history of tongue cancer treated by interstitial radiotherapy and had undergone a cytological examination of an ulcer on the right side of the tongue three weeks earlier because of suspected recurrence. The cytological examination result was class I with no malignant findings. Angioneurotic edema, so-called ''Quincke's edema'', associated with radiation-induced ulcer of the tongue, was diagnosed. Tranexamic acid, d-chlorpheniramine maleate, and epinephrine were administered. After six days, the tongue edema had almost disappeared. Laboratory examination revealed a low level of C1 inhibitor activity with normal levels of CH50, C1, C3, and C4 at the time of swelling. Hereditary angioneurotic edema with absence of hereditary trait was suspected based on the sudden edema attack and low level of C1 inhibitor activity. The C1 inhibitor activity returned to normal after disappearance of the tongue edema. (author)

Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

DEFF Research Database (Denmark)

Topakas, E.; Christakopoulos, Paul

2004-01-01

Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 1) produced activity at 82 nkat g(-1) dry substrate....... Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 degreesC with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5-8 and up...

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096.

Science.gov (United States)

Kumar, C Ganesh; Kamle, Avijeet; Kamal, Ahmed

2013-01-01

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE-1, AtFAE-2, and AtFAE-3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH-dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5-8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca(2+), K(+), and Mg(2+) stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k(cat) /K(m)) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF-MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE-1 belonged to type A while AtFAE-2 and AtFAE-3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

Science.gov (United States)

Varriale, Simona; Cerullo, Gabriella; Antonopoulou, Io; Christakopoulos, Paul; Rova, Ulrika; Tron, Thierry; Fauré, Régis; Jütten, Peter; Piechot, Alexander; Brás, Joana L A; Fontes, Carlos M G A; Faraco, Vincenza

2018-04-23

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Non-specific esterases in partly mineralized bovine enamel

DEFF Research Database (Denmark)

Moe, D; Kirkeby, S

1990-01-01

Activity for non-specific esterase was demonstrated in the matrix of developing bovine enamel with alpha-naphthyl acetate and 5-bromoindoxyl acetate as the esterase substrates. By use of high-performance liquid chromatography gel filtration, ion-exchange chromatography, and electrophoresis three...... esterases were shown to be present in the enamel matrix. The enzymes showed highest activity at pH 6.5-7.5. In sections a strong reaction was observed in the secretory ameloblasts. The esterases may be proteolytic enzymes that participate in the degradation of the matrix proteins....

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

Science.gov (United States)

2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

Science.gov (United States)

Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

2016-01-01

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

ELISA to measure neutralizing capacity of anti-C1-inhibitor antibodies in plasma of angioedema patients

NARCIS (Netherlands)

Engel, Ruchira; Rensink, Irma; Roem, Dorina; Brouwer, Mieke; Kalei, Asma; Perry, Dawn; Zeerleder, Sacha; Wouters, Diana; Hamann, Dörte

2015-01-01

Neutralizing autoantibodies (NAbs) against plasma serpin C1-inhibitor (C1-inh) are implicated in the rare disorder, acquired angioedema (AAE). There is insufficient understanding of the process of antibody formation and its correlation with disease progression and severity. We have developed an

Comparison of single and boosted protease inhibitor versus nonnucleoside reverse transcriptase inhibitor-containing cART regimens in antiretroviral-naïve patients starting cART after January 1, 2000

DEFF Research Database (Denmark)

Mocroft, A; Horban, A; Clumeck, N

2006-01-01

increase) response in antiretroviral-naïve patients starting either a single protease inhibitor (PI; n = 183), a ritonavir-boosted PI regimen (n = 197), or a nonnucleoside reverse transcriptase inhibitor (NNRTI)-based cART regimen (n = 447) after January 1, 2000, and the odds of lack of virologic...... or immunologic response at 3 years after starting cART. METHOD: Cox proportional hazards models and logistic regression. RESULTS: After adjustment, compared to patients taking an NNRTI-regimen, patients taking a single-PI regimen were significantly less likely to achieve a viral load (VL)

COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization

NARCIS (Netherlands)

Eldering, E.; Verpy, E.; Roem, D.; Meo, T.; Tosi, M.

1995-01-01

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from

Subcutaneous self-injections of C1 inhibitor: an effective and safe treatment in a patient with hereditary angio-oedema.

Science.gov (United States)

Weller, K; Krüger, R; Maurer, M; Magerl, M

2016-01-01

A 25-year-old woman presented to our clinic with a history of recurrent swelling and abdominal symptoms for > 20 years. The patient's father was similarly affected. The patient was diagnosed with hereditary angio-oedema (HAE) due to C1 inhibitor deficiency. This was initially managed with systemic androgens, but the symptoms of hyperandrogenism eventually became intolerable. Treatment with icatibant (an antagonist of bradykinin B2 receptors) was partially successful. We changed the therapy to prophylactic treatment with C1 inhibitor. Although the patient became completely symptom-free under this regimen, she found the repeated intravenous injections unacceptable. Therefore, we changed the route of administration to subcutaneous injections of C1 inhibitor 1000 U in 10 mL twice weekly, using a subcutaneous infusion kit. Since that time (December 2013), she has remained completely free of symptoms under this regimen. To our knowledge, this is the first report documenting the efficacy and safety of subcutaneous injections of C1 inhibitor in a patient with HAE. © 2015 British Association of Dermatologists.

Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

Science.gov (United States)

Zhang, Hao; Li, Fuchao; Chen, Huaxin; Zhao, Jin; Yan, Jinfei; Jiang, Peng; Li, Ronggui; Zhu, Baoli

2015-07-01

Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. ScsEst01 was successfully co-expressed in Escherichia coli BL21 (DE3) with chaperones (dnaK-dnaJ-grpE) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2+. The enzyme was characterized using p -nitrophenol butyrate as a substrate. ScsEst01 had the highest lipolytic activity at 35°C and pH 8.0, indicative of a meso-thermophilic alkaline esterase. ScsEst01 was thermostable at 20°C. The lipolytic activity of scsEst01 was strongly increased by Fe2+, Mn2+ and 1% Tween 80 or Tween 20.

Esterase inhibition by synergists in the western flower thrips Frankliniella occidentalis.

Science.gov (United States)

López-Soler, Neus; Cervera, Amelia; Quinto, Vicente; Abellán, Jaime; Bielza, Pablo; Martínez-Pardo, Rafael; Garcerá, Maria Dolores

2011-12-01

Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. In this work, only DEF shows true synergistic inhibition of WFT esterases. Copyright © 2011 Society of Chemical Industry.

Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

Directory of Open Access Journals (Sweden)

Moreno Galleni

2013-01-01

Full Text Available An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6–9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation. In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.

Gennemgang af en ny type hereditært angioødem med normal komplement C1-inhibitor

DEFF Research Database (Denmark)

Okholm-Hansen, Maria Bach; Winther, Anna Hillert; Fagerberg, Christina

2018-01-01

Hereditary angio-oedema (HAE) is a rare, potentially fatal disease characterized by recurrent swelling of skin and mucosa. Besides HAE with quantitative (type I) or qualitative (type II) deficiency of complement C1-inhibitor (C1-INH), a new subtype of HAE is now described with normal levels of C1...

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile

DEFF Research Database (Denmark)

Topakas, E.; Stamatis, H.; Biely, P.

2004-01-01

A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation......-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55-60 degreesC. The purified esterase was stable at the pH range 5.0-7.0. The enzyme retained 70% of activity after 7 h at 50 degreesC and lost 50% of its activity after 45 min at 55 degreesC and after 12 min at 60 degrees......C. Determination of k(cat)/K-m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2...

Esterase resistant to inactivation by heavy metals

KAUST Repository

El, Dorry Hamza

2014-09-25

EstATII is an esterase that a halotolerant, thermophilic and resistant to a spectrum of heavy metals including toxic concentration of metals. It was isolated from the lowest convective layer of the Atlantis II Red Sea brine pool. The Atlantis II brine pool is an extreme environment that possesses multiple harsh conditions such as; high temperature, salinity, pH and high concentration of metals, including toxic heavy metals. A fosmid metagenomic library using DNA isolated from the lowest convective layer this pool was used to identify EstATII. Polynucleotides encoding EstATII and similar esterases are disclosed and can be used to make EstATII. EstATII or compositions or apparatuses that contain it may be used in various processes employing lipases/esterases especially when these processes are performed under harsh conditions that inactivate other kinds of lipases or esterases.

A bacterial cocaine esterase protects against cocaine-induced epileptogenic activity and lethality.

Science.gov (United States)

Jutkiewicz, Emily M; Baladi, Michelle G; Cooper, Ziva D; Narasimhan, Diwahar; Sunahara, Roger K; Woods, James H

2009-09-01

Cocaine toxicity results in cardiovascular complications, seizures, and death and accounts for approximately 20% of drug-related emergency department visits every year. Presently, there are no treatments to eliminate the toxic effects of cocaine. The present study hypothesizes that a bacterial cocaine esterase with high catalytic efficiency would provide rapid and robust protection from cocaine-induced convulsions, epileptogenic activity, and lethality. Cocaine-induced paroxysmal activity and convulsions were evaluated in rats surgically implanted with radiotelemetry devices (N=6 per treatment group). Cocaine esterase was administered 1 minute after a lethal dose of cocaine or after cocaine-induced convulsions to determine the ability of the enzyme to prevent or reverse, respectively, the effects of cocaine. The cocaine esterase prevented all cocaine-induced electroencephalographic changes and lethality. This effect was specific for cocaine because the esterase did not prevent convulsions and death induced by a cocaine analog, (-)-2beta-carbomethoxy-3beta-phenyltropane. The esterase prevented lethality even after cocaine-induced convulsions occurred. In contrast, the short-acting benzodiazepine, midazolam, prevented cocaine-induced convulsions but not the lethal effects of cocaine. The data showed that cocaine esterase successfully degraded circulating cocaine to prevent lethality and that cocaine-induced convulsions alone are not responsible for the lethal effects of cocaine in this model. Therefore, further investigation into the use of cocaine esterase for treating cocaine overdose and its toxic effects is warranted.

Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis

NARCIS (Netherlands)

Droge, MJ; Bos, R; Quax, WJ

Carboxylesterase NP of Bacillus subtilis Thai 1-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene

Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp.

Science.gov (United States)

Oleas, Gabriela; Callegari, Eduardo; Sepúlveda, Romina; Eyzaguirre, Jaime

2017-04-18

The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10). Copyright © 2017 Elsevier Ltd. All rights reserved.

Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp.

Science.gov (United States)

Oleas, Gabriela; Callegari, Eduardo; Sepulveda, Romina; Eyzaguirre, Jaime

2016-01-01

The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation.

Solution Structure of a Novel C2-Symmetrical Bifunctional Bicyclic Inhibitor Based on SFTI-1

International Nuclear Information System (INIS)

Jaulent, Agnes M.; Brauer, Arnd B. E.; Matthews, Stephen J.; Leatherbarrow, Robin J.

2005-01-01

A novel bifunctional bicyclic inhibitor has been created that combines features both from the Bowman-Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), which has a compact bicyclic structure. The inhibitor was designed by fusing together a pair of reactive loops based on a sequence derived from SFTI-1 to create a backbone-cyclized disulfide-bridged 16-mer peptide. This peptide has two symmetrically spaced trypsin binding sites. Its synthesis and biological activity have been reported in a previous communication [Jaulent and Leatherbarrow, 2004, PEDS 17, 681]. In the present study we have examined the three-dimensional structure of the molecule. We find that the new inhibitor, which has a symmetrical 8-mer half-cystine CTKSIPP'I' motif repeated through a C 2 symmetry axis also shows a complete symmetry in its three-dimensional structure. Each of the two loops adopts the expected canonical conformation common to all BBIs as well as SFTI-1. We also find that the inhibitor displays a strong and unique structural identity, with a notable lack of minor conformational isomers that characterise most reactive site loop mimics examined to date as well as SFTI-1. This suggests that the presence of the additional cyclic loop acts to restrict conformational mobility and that the deliberate introduction of cyclic symmetry may offer a general route to locking the conformation of β-hairpin structures

Elucidating the Mechanism of Gain of Toxic Function From Mutant C1 Inhibitor Proteins in Hereditary Angioedema

Science.gov (United States)

2017-10-01

antibodies to 5 specifically blot wild-type C1INH in the pathologic polymers.. A FLAG tag was placed into the wild-type C1INH cDNA located immediately...resulted in decreased secretion of the 3x-FLAG-WT-C1INH when cotransfected with the mutant cDNA . This was an important confirmation of our...C1INH plus mutant C1INH cDNA in the presence or absence of a lactacystin, a proteasome inhibitor. As shown in figure 2, blocking degradation of

Structural analysis of thermostabilizing mutations of cocaine esterase

Energy Technology Data Exchange (ETDEWEB)

Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan; Ko, Mei-Chuan; Macdonald, Joanne; Tamburi, Patricia; Yoon, Dan; Landry, Donald M.; Woods, James H.; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K. (Michigan); (Columbia); (Kentucky)

2010-09-03

Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures ({tau}{sub 1/2} {approx} 13 min at 37 C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function.

Science.gov (United States)

Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-12-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the Skp-Cullin-F-box (SCF) complex. So far, no function has been ascribed to this domain. We find that the F-box of Rca1 is dispensable for APC/C-Fzr inhibition during G2. Nevertheless, we show that Rca1 has an additional function at the G1-S transition, which requires the F-box. Overexpression of Rca1 accelerates the G1-S transition in an F-box-dependent manner. Conversely, S-phase entry is delayed in cells in which endogenous Rca1 is replaced by a transgene lacking the F-box. We propose that Rca1 acts as an F-box protein in an as yet uncharacterized SCF complex, which promotes S-phase entry.

PRODUCTION AND CHARACTERIZATION OF AN ALKALOTHERMOSTABLE, ORGANIC SOLVENT TOLERANT AND SURFACTANT TOLERANT ESTERASE PRODUCED BY A THERMOPHILIC BACTERIUM GEOBACILLUS SP. AGP-04, ISOLATED FROM BAKRESHWAR HOT SPRING, INDIA

Directory of Open Access Journals (Sweden)

Amit Ghati

2013-10-01

Full Text Available A thermophilic bacteria, Geobacillus sp. AGP-04, isolated from Surya Kund hot spring, Bakreshwar, West Bengal, India was studied in terms of capability of tributyrin hydrolysis and characterization of its thermostable esterase activity using p-nitrophenyl butyrate (PNPB as substrate. The extracellular crude preparation was characterized in terms of pH and temperature optima and stability, organic solvent tolerance capacity and stability, substrate specificity, surfactant tolerance capacity, kinetic parameters and activation/inhibition behavior towards some metal ions and chemicals. Tributyrin agar assay exhibited that Geobacillus sp. AGP-04 secretes an extracellular esterase. The Vmax and Km values of the esterase were found to be 5099 U/Land 103.5µM, respectively in the presence of PNPB as substrate. The optimum temperature and pH, for Geobacillus sp. AGP-04 esterase was 60oC and 8.0, respectively. Although the enzyme activity was not significantly altered by incubating crude extract solution at 20-70oC for 1 hour, the enzyme activity was fully lost at 90oC for same incubation period. The pH stability profile showed that original crude esterase activity is stable at a broad range (pH 5.0-10.0. Moreover, the enzyme was highly organic solvent and surfactant tolerant. The effect of some chemical on crude esterase activity indicated that Geobacillus sp. AGP-04 produce an esterase which contains a serine residue in active site and for its activity -SH groups are essential. Besides, enzyme production was highly induced if fermentation medium contain polysaccharides and oil as carbon source.

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

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Koh Eunhee

2007-07-01

Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Esterase-lipase derived from Mucor miehei. 173.140... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by...

A comparison between activities for non-specific esterases and esterproteases

DEFF Research Database (Denmark)

Kirkeby, S; Moe, D

1988-01-01

Electrophoretic separation of non-specific esterases and esterproteases from kidney, lung, and liver have been carried out in polyacrylamide gels. By use of zone electrophoresis, isoelectric focusing, and 2-dimensional electrophoresis it was found that most of the esterprotease bands had the same...... localization in the gels as non-specific esterase bands. A number of esterase bands showed no activity towards the esterprotease substrates and a single kidney band possessed esterprotease activity only. Isozymes of the ES-6 and ES-9 zones showed sex dependent esterprotease reactions. In sections esterase...

Discovery and optimization of antibacterial AccC inhibitors

Energy Technology Data Exchange (ETDEWEB)

Cheng, Cliff C.; Shipps, Jr., Gerald W.; Yang, Zhiwei; Sun, Binyuan; Kawahata, Noriyuki; Soucy, Kyle A.; Soriano, Aileen; Orth, Peter; Xiao, Li; Mann, Paul; Black, Todd; (SPRI)

2010-09-17

The biotin carboxylase (AccC) is part of the multi-component bacterial acetyl coenzyme-A carboxylase (ACCase) and is essential for pathogen survival. We describe herein the affinity optimization of an initial hit to give 2-(2-chlorobenzylamino)-1-(cyclohexylmethyl)-1H-benzo[d]imidazole-5-carboxamide (1), which was identified using our proprietary Automated Ligand Identification System (ALIS). The X-ray co-crystal structure of 1 was solved and revealed several key interactions and opportunities for further optimization in the ATP site of AccC. Structure Based Drug Design (SBDD) and parallel synthetic approaches resulted in a novel series of AccC inhibitors, exemplified by (R)-2-(2-chlorobenzylamino)-1-(2,3-dihydro-1H-inden-1-yl)-1H-imidazo[4,5-b]pyridine-5-carboxamide (40). This compound is a potent and selective inhibitor of bacterial AccC with an IC{sub 50} of 20 nM and a MIC of 0.8 {micro}g/mL against a sensitized strain of Escherichia coli (HS294 E. coli).

Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors.

Directory of Open Access Journals (Sweden)

Elizabeth M Algar

Full Text Available SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi, Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

Larvicides and acetylcholinesterase inhibitors from Kalanchoe species; Atividades larvicida e anticolinesterasica de plantas do genero Kalanchoe

Energy Technology Data Exchange (ETDEWEB)

Trevisan, Maria Teresa Salles; Bezerra, Maria Zeneide Barbosa; Santiago, Gilvandete Maria Pinheiro; Feitosa, Chistiane Mendes [Ceara Univ., Fortaleza, CE (Brazil). Dept. de Quimica Organica e Inorganica]. E-mail: trevisan@ufc.br; Verpoorte, Robert [Leiden University, Leiden (Netherlands); Gorlaeus Laboratories, Leiden (Netherlands). Div. of Pharmacognosy; Braz Filho, Raimundo [Universidade Estadual do Norte Fluminense (UENF), Campos dos Goytacases, RJ (Brazil). Setor de Quimica de Produtos Naturais

2006-03-15

Acetylcholine esterase inhibitors are successy used to treat the symptoms of Alzheimer's disease. Extracts of three Kalanchoe species (K. brasiliensis, K. pinnata and K. gastonis-bornieri) showed acetylcholine esterase inhibitory effects and a toxic effect on Aedes aegypti larvae. Here we describe the bioassay guided fractionation of extracts of the most active extracts (K. brasiliensis) which resulted in the isolation of an active mixture of three flavonoids: 8-methoxyquercetin, 3,7-di-O-rhamnopyranoside and 8-methoxykaempferol-3,7-di-O-rhamnopyranoside. On TLC these flavonoids showed an acetylcholine esterase inhibitory effect. (author)

Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate ▿ †

Science.gov (United States)

Kabel, Mirjam A.; Yeoman, Carl J.; Han, Yejun; Dodd, Dylan; Abbas, Charles A.; de Bont, Jan A. M.; Morrison, Mark; Cann, Isaac K. O.; Mackie, Roderick I.

2011-01-01

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOSFA,Ac) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOSFA,Ac, a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23

Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from Geobacillus stearothermophilus

International Nuclear Information System (INIS)

Lansky, Shifra; Alalouf, Onit; Solomon, Vered; Alhassid, Anat; Govada, Lata; Chayan, Naomi E.; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

2013-01-01

The serine acetylxylan esterase from G. stearothermophilus (Axe2) has been crystallized in the tetragonal space group I422. Complete diffraction data sets have been measured for the selenomethionine derivative (SAD data, 1.70 Å resolution) and the wild-type enzyme (1.85 Å resolution) to be used for a full three-dimensional structural analysis of the Axe2 protein. Acetylxylan esterases are part of the hemi-cellulolytic system of many microorganisms which utilize plant biomass for growth. Xylans, which are polymeric sugars that constitute a significant part of the plant biomass, are usually substituted with acetyl side groups attached at position 2 or 3 of the xylose backbone units. Acetylxylan esterases hydrolyse the ester linkages of the xylan acetyl groups and thus improve the ability of main-chain hydrolysing enzymes to break down the sugar backbone units. As such, these enzymes play an important part in the hemi-cellulolytic utilization system of many microorganisms that use plant biomass for growth. Interest in the biochemical characterization and structural analysis of these enzymes stems from their numerous potential biotechnological applications. An acetylxylan esterase (Axe2) of this type from Geobacillus stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized. One of the crystal forms obtained (RB1) belonged to the tetragonal space group I422, with unit-cell parameters a = b = 110.2, c = 213.1 Å. A full diffraction data set was collected to 1.85 Å resolution from flash-cooled crystals of the wild-type enzyme at 100 K using synchrotron radiation. A selenomethionine derivative of Axe2 has also been prepared and crystallized for single-wavelength anomalous diffraction experiments. The crystals of the selenomethionine-derivatized Axe2 appeared to be isomorphous to those of the wild-type enzyme and enabled the measurement of a full 1.85 Å resolution diffraction data set at the selenium

Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

International Nuclear Information System (INIS)

Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J.; Goswami, Chandan

2014-01-01

Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys

Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

Energy Technology Data Exchange (ETDEWEB)

Kumar, Abhishek, E-mail: akumar@bot.uni-kiel.de [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Bhandari, Anita [Molecular Physiology, Zoological Institute, Christian-Albrechts-University at Kiel, Kiel (Germany); Sarde, Sandeep J. [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Goswami, Chandan [National Institute of Science Education and Research, Bhubaneswar, Orissa (India)

2014-07-18

Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

Enhanced biosurfactant production through cloning of three genes and role of esterase in biosurfactant release

Science.gov (United States)

2011-01-01

Background Biosurfactants have been reported to utilize a number of immiscible substrates and thereby facilitate the biodegradation of panoply of polyaromatic hydrocarbons. Olive oil is one such carbon source which has been explored by many researchers. However, studying the concomitant production of biosurfactant and esterase enzyme in the presence of olive oil in the Bacillus species and its recombinants is a relatively novel approach. Results Bacillus species isolated from endosulfan sprayed cashew plantation soil was cultivated on a number of hydrophobic substrates. Olive oil was found to be the best inducer of biosurfactant activity. The protein associated with the release of the biosurfactant was found to be an esterase. There was a twofold increase in the biosurfactant and esterase activities after the successful cloning of the biosurfactant genes from Bacillus subtilis SK320 into E.coli. Multiple sequence alignment showed regions of similarity and conserved sequences between biosurfactant and esterase genes, further confirming the symbiotic correlation between the two. Biosurfactants produced by Bacillus subtilis SK320 and recombinant strains BioS a, BioS b, BioS c were found to be effective emulsifiers, reducing the surface tension of water from 72 dynes/cm to as low as 30.7 dynes/cm. Conclusion The attributes of enhanced biosurfactant and esterase production by hyper-producing recombinant strains have many utilities from industrial viewpoint. This study for the first time has shown a possible association between biosurfactant production and esterase activity in any Bacillus species. Biosurfactant-esterase complex has been found to have powerful emulsification properties, which shows promising bioremediation, hydrocarbon biodegradation and pharmaceutical applications. PMID:21707984

Metagenomic mining of feruloyl esterases from termite enteric flora

CSIR Research Space (South Africa)

Rashamuse, K

2014-01-01

Full Text Available A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes...

Esterase polymorphism marking cultivars of Manihot esculenta, Crantz

Directory of Open Access Journals (Sweden)

Adriana Gazoli Resende

2004-07-01

Full Text Available Esterase isozymes were used to detected substrate-preference polymorphism in twenty cultivars of Manihot esculenta, and to show cultivar-specific variation of this species. A relatively complex extraction solution of proteins from leaves was needed to show a larger number of esterase isozymes. Similarity between cultivars from six groups ranged from 51 to 96%. The cultivars identified by the same name seemed to be biochemically different regarding esterase isozymes. Esterase isozyme electrophoretic patterns could, therefore, be used to discriminate the cultivars identified by the same name, and to monitor the vegetative propagation of cultivars maintained in the germplasm collection. In breeding strategies, isoesterase analysis could be used to avoid intercrossing between the similar genotypes.Isoenzimas esterases foram usadas no presente estudo, para detectar polimorfismos específicos para diferentes substratos em vinte cultivares de Manihot esculenta, e para mostrar variações específicas de cultivares nesta espécie. Os diferentes cultivares de M. esculenta tem sido mantidos na coleção de germoplasma do Departamento de Agronomia da Universidade Estadual de Maringá (Maringá, PR, e foram provenientes de cultivares tradicionais coletados nas regiões sudoeste e noroeste do Estado. Foi necessário a utilização de uma solução de extração de proteínas relativamente mais complexa, para evidenciar um maior número de isoenzimas esterases. A similaridade entre os cultivares variou de 51 a 96%. Cultivares identificados pelo mesmo nome parecem ser bioquimicamente diferentes para as isoenzimas esterases. Os padrões eletroforéticos das isoesterases podem, portanto, serem usados para discriminar os cultivares que são identificados pelo mesmo nome, e para monitorar a propagação vegetativa dos cultivares mantidos na coleção de germoplasma. A análise das isoesterases pode também ser usada para evitar cruzamentos entre genótipos mais

Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution.

Science.gov (United States)

Jiang, Huang; Zhang, Shaowei; Gao, Haofeng; Hu, Nan

2016-01-22

In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus, from India and its possible role in indoxacarb degradation

Directory of Open Access Journals (Sweden)

Shanivarsanthe Leelesh Ramya

2016-06-01

Full Text Available Abstract Diamondback moth (DBM, Plutella xylostella (Linnaeus, is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13 and adults (n = 12 of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%, followed by bacilli (15.4%. Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%, bacilli (16.7% and flavobacteria (16.7%. Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Effects of Model Salivary Esterases and MMP Inhibition on the Restoration's Marginal Integrity and Potential Degradative Contribution of Cariogenic Bacteria

Science.gov (United States)

Huang, Bo

Enzyme-catalyzed degradation of the restoration-tooth interface compromises interfacial integrity, thereby contributing to secondary caries, which is a major cause of resin-based restoration failure. It is hypothesized that in addition to salivary esterases, the cariogenic bacterium Streptococcus mutans has specific esterases that degrade the resin-dentin interface, releasing biodegradation by- products (BBPs) such as bis-hydroxy-propoxy-phenyl-propane (BisHPPP). In turn, BisHPPP affects S. mutans by stimulating the expression of esterases. Another hypothesis is that the biostability of the resin-dentin interface is affected by simulated salivary esterases, dentinal matrix metalloproteinase (MMP) inhibition, and restorative materials. To test the first hypothesis, putative esterase genes in S. mutans UA159 were identified, purified, and characterized. SMU_118c was identified as the dominant esterase in S. mutans UA159 and showed a similar hydrolytic activity profile to salivary esterases. BisHPPP upregulated expression of the SMU_118c gene and related protein in a concentration-dependent manner. This positive feedback process could accelerate the degradation of the restoration-tooth interface and lead to premature restoration failure. To test the second hypothesis, an in vitro model was established to evaluate the effects of salivary esterases, MMP inhibition and restorative materials on interfacial integrity. It was confirmed that interfacial integrity was compromised with time and was further deteriorated by simulated salivary esterases, as indicated by the greater depth of bacterial ingress and more bacterial biomass of biofilm along the interface. However, this process could be modulated by using different restorative materials and MMPs inhibition. This project elucidated the mechanistic interaction between oral bacteria and restorative materials and established a new, in vitro, and physiologically relevant model to assess the effect of material chemistry

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition.

Science.gov (United States)

Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2012-04-01

API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. 2012 AACR

The Lp_3561 and Lp_3562 enzymes support a functional divergence process in the lipase/esterase toolkit from Lactobacillus plantarum

Directory of Open Access Journals (Sweden)

Maria Esteban-Torres

2016-07-01

Full Text Available Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions.

Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

International Nuclear Information System (INIS)

Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

2008-01-01

The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2 1 2 1 2 1 . X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2 1 2 1 2 1 and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research

N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

Science.gov (United States)

Stavenhagen, Kathrin; Kayili, H Mehmet; Holst, Stephanie; Koeleman, Carolien A M; Engel, Ruchira; Wouters, Diana; Zeerleder, Sacha; Salih, Bekir; Wuhrer, Manfred

2018-06-01

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N - and O -glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O -glycosylated N-terminal region. Five novel and five known O -glycosylation sites were identified, carrying mainly core1-type O -glycans. In addition, we detected a heavily O -glycosylated portion spanning from Thr 82 -Ser 121 with up to 16 O -glycans attached. Likewise, all known six N -glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N -glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Feruloyl esterase from Aspergillus clavatus improves xylan hydrolysis of sugarcane bagasse

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Dyoni M. de Oliveira

2016-12-01

Full Text Available Feruloyl esterase is a subclass of carboxylic acid esterases with the capacity to release ferulic acid and other cinnamic acids from plant cell walls and synthetic substrates. Feruloyl esterases act synergistically with xylanases removing ferulic acid residues esterified to arabinoxylans. Feruloyl esterase type D from Aspergillus clavatus (AcFAE was expressed in Escherichia coli, purified, and applied with a commercial xylanase consortium (Novozymes for hydrolysis of sugarcane bagasse. Feruloyl esterase plus xylanase increased 5.13-fold the releasing of ferulic acid from sugarcane bagasse. Removal of only 7.7% of ferulic acid content by AcFAE increased 97.3% the sugarcane bagasse hydrolysis by xylanase. These data support the use of AcFAE as an interesting adjuvant enzyme to improve lignocellulose digestion and biotechnological tool for biorefineries.

Potential physiological role of plant glycosidase inhibitors

DEFF Research Database (Denmark)

Bellincampi, D.; Carmadella, L.; Delcour, J.A.

2004-01-01

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens...... and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role...... of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological...

Inhibitors of the alpha-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons.

Science.gov (United States)

Santos, Sónia Sá; Gibson, Gary E; Cooper, Arthur J L; Denton, Travis T; Thompson, Charles M; Bunik, Victoria I; Alves, Paula M; Sonnewald, Ursula

2006-02-15

Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-13C]glucose and [U-13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-13C]glutamate from [1-13C]glucose, a reduction in label in glutamate derived from [1-13C]glucose/[U-13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

Preliminary X-ray Study of Naproxen Esterase from Bacillus subtilis

NARCIS (Netherlands)

van der Laan, Jan; Teplyakov, A.V.; Lammers, A.A.; Dijkstra, B.W.

1993-01-01

Single crystals of naproxen esterase from Bacillus subtilis have been obtained from PEG6000 solutions at pH 8.0 by liquid-liquid diffusion while applying a temperature gradient from 4°C to room temperature over a period of four weeks. The crystals belong to the trigonal space group P3121 or P3221

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

Science.gov (United States)

Martin del Campo, Martha; Camacho, Rosa M; Mateos-Díaz, Juan C; Müller-Santos, Marcelo; Córdova, Jesus; Rodríguez, Jorge A

2015-11-01

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer.

Science.gov (United States)

Afifi, A F; Fawzi, E M; Foaad, M A

2002-01-01

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.

Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

Energy Technology Data Exchange (ETDEWEB)

Wood, S. J. [Biosciences Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Li, X.-L.; Cotta, M. A. [Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois 61604 (United States); Biely, P. [Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava (Slovakia); Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R., E-mail: rajp@anl.gov [Biosciences Division, Argonne National Laboratory, Argonne, IL 60439 (United States)

2008-04-01

The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

Atividades larvicida e anticolinesterásica de plantas do gênero Kalanchoe Larvicides and acetylcholinesterase inhibitors from Kalanchoe species

Directory of Open Access Journals (Sweden)

Maria Teresa Salles Trevisan

2006-06-01

Full Text Available Acetylcholine esterase inhibitors are successfully used to treat the symptoms of Alzheimer's disease. Extracts of three Kalanchoe species (K. brasiliensis, K. pinnata and K. gastonis-bornieri showed acetylcholine esterase inhibitory effects and a toxic effect on Aedes aegypti larvae. Here we describe the bioassay guided fractionation of extracts of the most active extracts (K. brasiliensis which resulted in the isolation of an active mixture of three flavonoids: 8-methoxyquercetin, 3,7-di-O-rhamnopyranoside and 8-methoxykaempferol-3,7-di-O-rhamnopyranoside. On TLC these flavonoids showed an acetylcholine esterase inhibitory effect.

Recent Developments of C-Aryl Glucoside SGLT2 Inhibitors.

Science.gov (United States)

Zhang, Yang; Liu, Zhao-Peng

2016-01-01

Sodium-glucose cotransporter 2 (SGLT2) is almost exclusively expressed in the proximal renal tubules. It is responsible for about 90% of the glucose reabsorption from tubular fluid. Selective inhibition of SGLT2 is expected to favor in the normalization of plasma glucose levels in T2DM patients through the prevention of renal glucose reabsorption and the promotion of glucose excretion from urine. Selective SGLT2 inhibitors have the merits to minimize the gastrointestinal side effects associated with SGLT1 inhibition, and selective SGLT2 inhibition may have a low risk of hypoglycemia. Since the C-aryl glucosides are metabolically more stable than the O-glucosides, numerous efforts have been made in the development of potent and selective C-aryl glucoside SGLT2 inhibitors, and a number of them are now used as anti-diabetes drugs in clinic or at various stages of clinical developments. Based on their structural features, in this review, these SGLT2 inhibitors are classified as three types: the phenyl/arylmethylphenyl C-glucosides, with an emphasis on the modifications on the proximal and/or the distal phenyl ring, and the spacer; the heteroarylmethylphenyl Cglucosides, with a replacement of the distal phenyl ring by a heterocycle like pyridazine, pyrimidine, thiophene and benzothiophene, thiazole, 1,3,4-thiadiazole, and triazolopyridinone; and the glucose-modified Caryl glucosides, including the glucose C-1 derived O-spiroketals, C-4 gem-difluoro analogues, C-5 and C-6 modified derivatives, dioxa-bicyclo[3.2.1]octane bridged ketals, the thioglucosides, and carbasugars. The structure-activity relationships (SARs) of each type along with their inhibitory potency against human SGLT2 and selectivity over human SGLT1 are discussed.

Esterase activity in the guinea pig thyroid under normal and pathological conditions (vitamin A deficiency) with special regard to cyst-like structures

DEFF Research Database (Denmark)

Kirkeby, S

1977-01-01

By use of different activators and inhibitors, TOCP(tri-o-cresyl phosphate), PCMB (parachloromercury benzoate), NiCl2, Pb(NO3)2, HgCl2, Hg(NO3)2, eserine and sodium taurocholate, it is shown that the esterase in the cyst cells and in group I cells of the guinea pig thyroid probably are A...

International consensus on the diagnosis and management of pediatric patients with hereditary angioedema with C1 inhibitor deficiency.

Science.gov (United States)

Farkas, H; Martinez-Saguer, I; Bork, K; Bowen, T; Craig, T; Frank, M; Germenis, A E; Grumach, A S; Luczay, A; Varga, L; Zanichelli, A

2017-02-01

The consensus documents published to date on hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) have focused on adult patients. Many of the previous recommendations have not been adapted to pediatric patients. We intended to produce consensus recommendations for the diagnosis and management of pediatric patients with C1-INH-HAE. During an expert panel meeting that took place during the 9th C1 Inhibitor Deficiency Workshop in Budapest, 2015 (www.haenet.hu), pediatric data were presented and discussed and a consensus was developed by voting. The symptoms of C1-INH-HAE often present in childhood. Differential diagnosis can be difficult as abdominal pain is common in pediatric C1-INH-HAE, but also commonly occurs in the general pediatric population. The early onset of symptoms may predict a more severe subsequent course of the disease. Before the age of 1 year, C1-INH levels may be lower than in adults; therefore, it is advisable to confirm the diagnosis after the age of one year. All neonates/infants with an affected C1-INH-HAE family member should be screened for C1-INH deficiency. Pediatric patients should always carry a C1-INH-HAE information card and medicine for emergency use. The regulatory approval status of the drugs for prophylaxis and for acute treatment is different in each country. Plasma-derived C1-INH, recombinant C1-INH, and ecallantide are the only agents licensed for the acute treatment of pediatric patients. Clinical trials are underway with additional drugs. It is recommended to follow up patients in an HAE comprehensive care center. The pediatric-focused international consensus for the diagnosis and management of C1-INH-HAE patients was created. © 2016 The Authors. Allergy Published by John Wiley & Sons Ltd.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation.

Science.gov (United States)

Ramya, Shanivarsanthe Leelesh; Venkatesan, Thiruvengadam; Srinivasa Murthy, Kottilingam; Jalali, Sushil Kumar; Verghese, Abraham

2016-01-01

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32μmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26μmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Binding of the Inhibitor Protein IF1 to Bovine F1-ATPase

Science.gov (United States)

Bason, John V.; Runswick, Michael J.; Fearnley, Ian M.; Walker, John E.

2011-01-01

In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the α-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF1 and the C-terminal domains of the βDP-subunit and βTP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the βDP-subunit. Several conserved charged amino acids in the long α-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme. PMID:21192948

Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure in Indian patients.

Science.gov (United States)

Neogi, Ujjwal; Rao, Shwetha D; Bontell, Irene; Verheyen, Jens; Rao, Vasudev R; Gore, Sagar C; Soni, Neelesh; Shet, Anita; Schülter, Eugen; Ekstrand, Maria L; Wondwossen, Amogne; Kaiser, Rolf; Madhusudhan, Mallur S; Prasad, Vinayaka R; Sonnerborg, Anders

2014-09-24

A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region, which appeared in protease inhibitor failure Indian HIV-1C sequences (odds ratio=17.1, P < 0.001) but was naturally present in half of untreated Ethiopian HIV-1C sequences. The insertion is predicted to restore ALIX-mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of the insertion needs to be evaluated in HIV-1C dominating regions wherein the use of protease inhibitor drugs are being scaled up.

Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

Energy Technology Data Exchange (ETDEWEB)

Hiraki, Toshiki [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Shibayama, Naoya [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Yoon, Young-Ho [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Yun, Kyung-Mook [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Hamamoto, Toshiro [Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Tame, Jeremy R. H.; Park, Sam-Yong, E-mail: park@tsurumi.yokohama-cu.ac.jp [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan)

2007-09-01

Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S.

Science.gov (United States)

Luo, Xiangwen; Zhang, Deyong; Zhou, Xuguo; Du, Jiao; Zhang, Songbai; Liu, Yong

2018-05-09

Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30-46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l -1 and 0.918 ± 0.025 U·µg -1 , respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

Esterase Isoenzyme Profiles in Acute and Chronic Leukemias.

Science.gov (United States)

Drexler, H G; Gignac, S M; Hoffbrand, A V; Minowada, J

1991-01-01

Using isoelectric focusing (IEF) a number of carboxylic esterase isoenzymes (EC 3.1.1.1) with isoelectric points between pH 4.5-8.0 can be separated. One particular isoenzyme with an isoelectric point at about pH 6.0, the Mono-band, can be selectively and completely inhibited by sodium fluoride; this isoenzyme comprises a number of closely related subcomponents and may appear in more than one band on the gel. We analyzed the expression of typical esterase isoenzyme patterns in cells from a large panel of leukemias which were tested under identical conditions by IEF on horizontal thin-layer polyacrylamide gels with an ampholyte of pH 2-11. The 442 cases of acute and chronic myeloid and lymphoid leukemia (AML/AMMoL, CML/CMML, ALL, CLL) were classified according to clinical, morpho-cytochemical and immunophenotyping criteria. While bands between pH 4.5-5.5 appeared not to be specific for lineage or stage of differentiation, isoenzymes between pH 6.6-7.7 provided information on the type of leukemia involved. Seven typical isoenzyme patterns termed Mono1/Mono2 (fo monocyte-associated), My1/My2 (myeloid), Lym1/Lym2 (lymphoid) and Und (undifferentiated) could be discerned. Lym and Und patterns are characterized by fewer bands with a weaker staining intensity than Mono and My patterns. Nearly all cases of lymphoid leukemias (acute and chronic) expressed only Lym or Und esterase isoenzyme patterns, but no Mono or My patterns. Cases of acute or chronic myeloid and (myelo)monocytic leukemia showed strong isoenzyme staining displaying predominantly Mono or My isoenzyme patterns. The isoenzyme patterns found in CML in lymphoid or myeloid blast crisis corresponded to those seen in the respective acute leukemias, ALL or AML. The Mono-band was found in most cases of leukemias with monocytic elements (AMMoL 80%, CML 44%, CMML 100%), in the occasional case of CML-myeloid blast crisis or AML, but in none of the cases of ALL or CLL. This isoenzyme is a distinctive, specific marker for

Health-Related Quality of Life with Subcutaneous C1-Inhibitor for Prevention of Attacks of Hereditary Angioedema.

Science.gov (United States)

Lumry, William R; Craig, Timothy; Zuraw, Bruce; Longhurst, Hilary; Baker, James; Li, H Henry; Bernstein, Jonathan A; Anderson, John; Riedl, Marc A; Manning, Michael E; Keith, Paul K; Levy, Donald S; Caballero, Teresa; Banerji, Aleena; Gower, Richard G; Farkas, Henriette; Lawo, John-Philip; Pragst, Ingo; Machnig, Thomas; Watson, Douglas J

2018-01-31

Hereditary angioedema with C1-inhibitor deficiency (C1-INH-HAE) impairs health-related quality of life (HRQoL). The objective of this study was to assess HRQoL outcomes in patients self-administering subcutaneous C1-INH (C1-INH[SC]; HAEGARDA) for routine prevention of HAE attacks. Post hoc analysis of data from the placebo-controlled, crossover phase III COMPACT study (Clinical Studies for Optimal Management of Preventing Angioedema with Low-Volume Subcutaneous C1-Inhibitor Replacement Therapy). Ninety patients with C1-INH-HAE were randomized to 1 of 4 treatment sequences: C1-INH(SC) 40 or 60 IU/kg twice weekly for 16 weeks, preceded or followed by 16 weeks of twice weekly placebo injections. All HAE attacks were treated with open-label on-demand treatment as necessary. HRQoL assessments at week 14 (last visit) included the European Quality of Life-5 Dimensions Questionnaire (EQ-5D-3L), the Hospital Anxiety and Depression Scale (HADS), the Work Productivity and Activity Impairment Questionnaire (WPAI), and the Treatment Satisfaction Questionnaire for Medication (TSQM). Compared with placebo (on-demand treatment alone), treatment with twice weekly C1-INH(SC) (both doses combined) was associated with better EQ-5D visual analog scale general health, less HADS anxiety, less WPAI presenteeism, work productivity loss, and activity impairment, and greater TSQM effectiveness and overall treatment satisfaction. More patients self-reported a "good/excellent" response during routine prevention with C1-INH(SC) compared with on-demand only (placebo prophylaxis) management. For each HRQoL measure, a greater proportion of patients had a clinically meaningful improvement during C1-INH(SC) treatment compared with placebo. In patients with frequent HAE attacks, a treatment strategy of routine prevention with self-administered twice weekly C1-INH(SC) had a greater impact on improving multiple HAE-related HRQoL impairments, most notably anxiety and work productivity, compared with on

Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

Science.gov (United States)

Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

2017-03-15

Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

Science.gov (United States)

Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

2015-12-01

Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Plasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database ( http://hivdb.stanford.edu ) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates. All subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions. Although the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species

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Tuffery Pierre

2009-12-01

Full Text Available Abstract Background Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. Results We identified the gene encoding esterase B as the acetyl-esterase gene (aes using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Conclusion Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

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Pichapak Sriyapai

2015-06-01

Full Text Available A thermostable esterase gene (hydS14 was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG and catalytic triad (Ser88-Asp208-His235 of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases, has three conserved regions, and contains the novel motif (GY(FSLG, which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14 was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6, displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1. In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

Design and synthesis of 3,3'-biscoumarin-based c-Met inhibitors.

Science.gov (United States)

Xu, Jimin; Ai, Jing; Liu, Sheng; Peng, Xia; Yu, Linqian; Geng, Meiyu; Nan, Fajun

2014-06-14

A library of biscoumarin-based c-Met inhibitors was synthesized, based on optimization of 3,3'-biscoumarin hit 3, which was identified as a non-ATP competitive inhibitor of c-Met from a diverse library of coumarin derivatives. Among these compounds, 38 and 40 not only showed potent enzyme activities with IC50 values of 107 nM and 30 nM, respectively, but also inhibited c-Met phosphorylation in BaF3/TPR-Met and EBC-1 cells.

Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

Science.gov (United States)

Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

2017-09-01

A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål).

Science.gov (United States)

Vontas, J G; Small, G J; Hemingway, J

2000-12-01

Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.

Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae

Energy Technology Data Exchange (ETDEWEB)

Aparna, Gudlur; Chatterjee, Avradip; Jha, Gopaljee; Sonti, Ramesh V.; Sankaranarayanan, Rajan, E-mail: sankar@ccmb.res.in [Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007 (India)

2007-08-01

The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported. Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.

Geranyl acetate esterase controls and regulates the level of geraniol in lemongrass (Cymbopogon flexuosus Nees ex Steud.) mutant cv. GRL-1 leaves.

Science.gov (United States)

Ganjewala, Deepak; Luthra, Rajesh

2009-01-01

Essential oil isolated from lemongrass (Cymbopogon flexuosus) mutant cv. GRL-1 leaves is mainly composed of geraniol (G) and geranyl acetate (GA). The proportion of G and GA markedly fluctuates during leaf development. The proportions of GA and G in the essential oil recorded at day 10 after leaf emergence were approximately 59% and approximately 33% respectively. However, the level of GA went down from approximately 59 to approximately 3% whereas the level of G rose from approximately 33 to approximately 91% during the leaf growth period from day 10 to day 50. However, the decline in the level of GA was most pronounced in the early (day 10 to day 30) stage of leaf growth. The trend of changes in the proportion of GA and G has clearly indicated the role of an esterase that must be involved in the conversion of GA to G during leaf development. We isolated an esterase from leaves of different ages that converts GA into G and has been given the name geranyl acetate esterase (GAE). The GAE activity markedly varied during the leaf development cycle; it was closely correlated with the monoterpene (GA and G) composition throughout leaf development. GAE appeared as several isoenzymes but only three (GAE-I, GAE-II, and GAE-III) of them had significant GA cleaving activity. The GAE isoenzymes pattern was greatly influenced by the leaf developmental stages and so their GA cleaving activities. Like the GAE activity, GAE isoenzyme patterns were also found to be consistent with the monoterpene (GA and G) composition. GAE had an optimum pH at 8.5 and temperature at 30 degrees C. Besides GAE, a compound with phosphatase activity capable of hydrolyzing geranyl diphosphate (GPP) to produce geraniol has also been isolated.

Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals

DEFF Research Database (Denmark)

Andreasen, Mette Findal; Kroon, P A; Williamson, G

2001-01-01

and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary...... hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained...... from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye...

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

International Nuclear Information System (INIS)

Vose, Sarah C.; Fujioka, Kazutoshi; Gulevich, Alex G.; Lin, Amy Y.; Holland, Nina T.; Casida, John E.

2008-01-01

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action

A novel small molecule inhibitor of hepatitis C virus entry.

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Carl J Baldick

Full Text Available Small molecule inhibitors of hepatitis C virus (HCV are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc, blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Use of a C1 Inhibitor Concentrate in Adults ≥65 Years of Age with Hereditary Angioedema

DEFF Research Database (Denmark)

Bygum, Anette; Martinez-Saguer, Inmaculada; Bas, Murat

2016-01-01

BACKGROUND: Treatment of hereditary angioedema (HAE) in 'older adults' (those aged ≥65 years) has not been well studied. The international Berinert Patient Registry collected data on the use of intravenous plasma-derived, pasteurized, nanofiltered C1-inhibitor concentrate (pnfC1-INH; Berinert......(®)/CSL Behring) in patients of any age, including many older adults. METHODS: This observational registry, conducted from 2010 to 2014 at 30 US and seven European sites, gathered prospective (post-enrollment) and retrospective (pre-enrollment) usage and adverse event (AE) data on subjects treated with pnfC1-INH....... RESULTS: The registry documented 1701 pnfC1-INH infusions in 27 older adults. A total of 1511 HAE attacks treated with pnfC1-INH administration were reported among 25 of the 27 (92.6 %) older adults. Among the older adults, mean (standard deviation [SD]) (8.8 [4.1] IU/kg) and median (6.4 IU/kg) pnfC1-INH...

Determination of activities of human carbonic anhydrase II inhibitors ...

African Journals Online (AJOL)

Purpose: To evaluate the activities of new curcumin analogs as carbonic anhydrase II (CA-II) inhibitor. Methods: Carbonic anhydrase II (CA-II) inhibition was determined by each ligand capability to inhibit the esterase activity of CA-II using 4-NPA as a substrate in 96-well plates. Dimethyl sulfoxide was used to dissolve each ...

Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst.

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Jose L S Lopes

Full Text Available Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2

Energy Technology Data Exchange (ETDEWEB)

Labroli, Marc; Paruch, Kamil; Dwyer, Michael P.; Alvarez, Carmen; Keertikar, Kartik; Poker, Cory; Rossman, Randall; Duca, Jose S.; Fischmann, Thierry O.; Madison, Vincent; Parry, David; Davis, Nicole; Seghezzi, Wolfgang; Wiswell, Derek; Guzi, Timothy J. [Merck

2013-11-20

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

Protein C Inhibitor-A Novel Antimicrobial Agent

NARCIS (Netherlands)

Malmström, E.; Mörgelin, M.; Malmsten, M.; Johansson, L.; Norrby-Teglund, A.; Shannon, O.; Schmidtchen, A.; Meijers, J.C.M.; Herwald, H.

2009-01-01

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which

Interactions between resin monomers and commercial composite resins with human saliva derived esterases.

Science.gov (United States)

Jaffer, F; Finer, Y; Santerre, J P

2002-04-01

Cholesterol esterase (CE) and pseudocholinesterase (PCE) have been reported to degrade commercial and model composite resins containing bisphenylglycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) or the latter in combination with urethane modified BisGMA monomer systems. In addition, human saliva has been shown to contain esterase like activities similar to CE and PCE. Hence, it was the aim of the current study to determine to what extent human saliva could degrade two common commercial composite resins (Z250 from 3M Inc. and Spectrum TPH from L.D. Caulk) which contain the above monomer systems. Saliva samples from different volunteers were collected, processed, pooled, and freeze-dried. TEGDMA and BisGMA monomers were incubated with human saliva derived esterase activity (HSDEA) and their respective hydrolysis was monitored using high performance liquid chromatography (HPLC). Both monomers were completely hydrolyzed within 25 h by HSDEA. Photopolymerized composites were incubated with buffer or human saliva (pH 7.0 and 37 C) for 2, 8 and 16 days. The incubation solutions were analyzed using HPLC and mass spectrometry. Surface morphology characterization was carried out using scanning electron microscopy. Upon biodegradation, the Z250 composite yielded higher amounts of BisGMA and TEGDMA related products relative to the TPH composite. However, there were higher amounts of ethoxylated bis-phenol A released from the TPH material. In terms of total mass of products released, human saliva demonstrated a greater ability to degrade Z250. In summary, HSDEA has been shown to contain esterase activities that can readily catalyze the biodegradation of current commercial composite resins.

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

Science.gov (United States)

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Dual mTORC1/C2 inhibitors suppress cellular geroconversion (a senescence program).

Science.gov (United States)

Leontieva, Olga V; Demidenko, Zoya N; Blagosklonny, Mikhail V

2015-09-15

In proliferating cells, mTOR is active and promotes cell growth. When the cell cycle is arrested, then mTOR converts reversible arrest to senescence (geroconversion). Rapamycin and other rapalogs suppress geroconversion, maintaining quiescence instead. Here we showed that ATP-competitive kinase inhibitors (Torin1 and PP242), which inhibit both mTORC1 and TORC2, also suppressed geroconversion. Despite inhibition of proliferation (in proliferating cells), mTOR inhibitors preserved re-proliferative potential (RP) in arrested cells. In p21-arrested cells, Torin 1 and PP242 detectably suppressed geroconversion at concentrations as low as 1-3 nM and 10-30 nM, reaching maximal gerosuppression at 30 nM and 300 nM, respectively. Near-maximal gerosuppression coincided with inhibition of p-S6K(T389) and p-S6(S235/236). Dual mTOR inhibitors prevented senescent morphology and hypertrophy. Our study warrants investigation into whether low doses of dual mTOR inhibitors will prolong animal life span and delay age-related diseases. A new class of potential anti-aging drugs can be envisioned.

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

Science.gov (United States)

Mangas, I; Vilanova, E; Benabent, M; Estévez, J

2014-02-10

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Esterases in striated muscle from mice with the Chediak-Higashi syndrome

DEFF Research Database (Denmark)

Kirkeby, S; Moe, D

1981-01-01

In this paper a localized strong reaction for non-specific esterase forming cylindric structures is described within skeletal muscle fibres from the beige mouse. It seems from zymograms and protein electrophoresis that this esterase is membrane bound, highly reactive and present in rather small...

C1 Inhibitor in Acute Antibody-Mediated Rejection Nonresponsive to Conventional Therapy in Kidney Transplant Recipients: A Pilot Study.

Science.gov (United States)

Viglietti, D; Gosset, C; Loupy, A; Deville, L; Verine, J; Zeevi, A; Glotz, D; Lefaucheur, C

2016-05-01

Complement inhibitors have not been thoroughly evaluated in the treatment of acute antibody-mediated rejection (ABMR). We performed a prospective, single-arm pilot study to investigate the potential effects and safety of C1 inhibitor (C1-INH) Berinert added to high-dose intravenous immunoglobulin (IVIG) for the treatment of acute ABMR that is nonresponsive to conventional therapy. Kidney recipients with nonresponsive active ABMR and acute allograft dysfunction were enrolled between April 2013 and July 2014 and received C1-INH and IVIG for 6 months (six patients). The primary end point was the change in eGFR at 6 months after inclusion (M+6). Secondary end points included the changes in histology and DSA characteristics and adverse events as evaluated at M+6. All patients showed an improvement in eGFR between inclusion and M+6: from 38.7 ± 17.9 to 45.2 ± 21.3 mL/min/1.73 m(2) (p = 0.0277). There was no change in histological features, except a decrease in the C4d deposition rate from 5/6 to 1/6 (p = 0.0455). There was a change in DSA C1q status from 6/6 to 1/6 positive (p = 0.0253). One deep venous thrombosis was observed. In a secondary analysis, C1-INH patients were compared with a similar historical control group (21 patients). C1-INH added to IVIG is safe and may improve allograft function in kidney recipients with nonresponsive acute ABMR. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

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Renaud Berlemont

2013-03-01

Full Text Available In order to isolate novel organic solvent-tolerant (OST lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1 contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04. Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4 compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST, se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un

Fitness differences due to allelic variation at Esterase-4 locus in ...

Indian Academy of Sciences (India)

KAVITA KRISHNAMOORTI

2017-08-31

Aug 31, 2017 ... Keywords. esterases; null allele; reproductive fitness; natural selection; Drosophila ananassae. .... cific substrate (1-naphthylacetate AR) and stain (fast blue. RR). On the ... transferred to fresh food vials and eggs were counted.

Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

Energy Technology Data Exchange (ETDEWEB)

Byun, Jung-Sue [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of); Rhee, Jin-Kyu [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Oh, Jong-Won [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

2006-02-01

Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method. EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4–C6 at a temperature of 303–363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est{sub pc}-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 Å resolution from an EstE1 crystal; the crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 73.71, c = 234.23 Å. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient V{sub M} is calculated to be 2.2 Å{sup 3} Da{sup −1} and the solvent content is 44.1%.

A Novel Halotolerant Thermoalkaliphilic Esterase from Marine Bacterium Erythrobacter seohaensis SW-135

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Ying-Yi Huo

2017-11-01

Full Text Available A novel esterase gene, e69, was cloned from Erythrobacter seohaensis SW-135, which was isolated from a tidal flat sediment of the Yellow Sea in Korea. This gene is 825 bp in length and codes for a 29.54 kDa protein containing 274 amino acids. Phylogenetic analysis showed that E69 is a new member of the bacterial lipolytic enzyme family IV. This enzyme exhibited the highest level of activity toward p-nitrophenyl (NP butyrate but little or no activity toward the other p-NP esters tested. The optimum temperature and pH of the catalytic activity of E69 were 60°C and pH 10.5, respectively. The enzyme exhibited stable activity over a wide range of alkaline pH values (7.5–9.5. In addition, E69 was found to be a halotolerant esterase as it exhibited the highest hydrolytic activity in the presence of 0.5 M NaCl and was still active in the presence of 3 M NaCl. Moreover, it possessed some degree of tolerance to Triton X-100 and several organic solvents. Through homology modeling and comparison with other esterases, it was suggested that the absence of the cap domain and its narrow substrate-binding pocket might be responsible for its narrow substrate specificity. Sequence and structural analysis results suggested that its high ratio of negatively to positively charged residues, large hydrophobic surface area, and negative electrostatic potential on the surface may be responsible for its alkaline adaptation. The results of this study provide insight into marine alkaliphilic esterases, and the unique properties of E69 make it a promising candidate as a biocatalyst for industrial applications.

In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.

Science.gov (United States)

Herholz, K; Bauer, B; Wienhard, K; Kracht, L; Mielke, R; Lenz, M O; Strotmann, T; Heiss, W D

2000-01-01

Memory and attention are cognitive functions that depend heavily on the cholinergic system. Local activity of acetylcholine esterase (AChE) is an indicator of its integrity. Using a recently developed tracer for positron emission tomography (PET), C-11-labeled N-methyl-4-piperidyl-acetate (C11-MP4A), we measured regional AChE activity in 4 non-demented subjects, 4 patients with dementia of Alzheimer type (DAT) and 1 patient with senile dementia of Lewy body type (SDLT), and compared the findings with measurements of blood flow (CBF) and glucose metabolism (CMRGlc). Initial tracer extraction was closely related to CBF. AChE activity was reduced significantly in all brain regions in demented subjects, whereas reduction of CMRGlc and CBF was more limited to temporo-parietal association areas. AChE activity in SDLT was in the lower range of values in DAT. Our results indicate that, compared to non-demented controls, there is a global reduction of cortical AChE activity in dementia. Dementia, cholinergic system, acetylcholine esterase, positron emission tomography, cerebral blood flow, cerebral glucose metabolism.

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

Science.gov (United States)

Chen, Qi; Luan, Zheng-Jiao; Yu, Hui-Lei; Cheng, Xiaolin; Xu, Jian-He

2015-11-01

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications. Copyright © 2015 Elsevier Inc. All rights reserved.

[Determination of drug resistance mutations of NS3 inhibitors in chronic hepatitis C patients infected with genotype 1].

Science.gov (United States)

Şanlıdağ, Tamer; Sayan, Murat; Akçalı, Sinem; Kasap, Elmas; Buran, Tahir; Arıkan, Ayşe

2017-04-01

Direct-acting antiviral agents (DAA) such as NS3 protease inhibitors is the first class of drugs used for chronic hepatitis C (CHC) treatment. NS3 inhibitors (PI) with low genetic barrier have been approved to be used in the CHC genotype 1 infections, and in the treatment of compensated liver disease including cirrhosis together with pegile interferon and ribavirin. Consequently, the development of drug resistance during DAA treatment of CHC is a major problem. NS3 resistant variants can be detected before treatment as they can occurnaturally. The aim of this study was to investigate new and old generation NS3 inhibitors resistance mutations before DAA treatment in hepatitis C virus (HCV) that were isolated from CHC. The present study was conducted in 2015 and included 97 naive DAA patients infected with HCV genotype 1, who were diagnosed in Manisa and Kocaeli cities of Turkey. Magnetic particle based HCV RNA extraction and than RNA detection and quantification were performed using commercial real-time PCR assay QIASypmhony + Rotorgene Q/ArtusHCV QS-RGQ and COBAS Ampliprep/COBAS TaqMan HCV Tests. HCV NS3 viral protease genome region was amplified with PCR and mutation analysis was performed by Sanger dideoxy sequencing technique of NS3 protease codons (codon 32-185). HCV NS3 protease inhibitors; asunaprevir, boceprevir, faldaprevir, grazoprevir, pariteprevir, simeprevir and telaprevir were analysed for resistant mutations by Geno2pheno-HCV resistance tool. HCV was genotyped in all patients and 88 patients (n= 88/97, 91%) had genotype 1. Eight (n= 8/97, 8.2%) and 80 (n= 80/97, 82.4%) HCC patients were subgenotyped as 1a and 1b, respectively. Many aminoacid substitutions and resistance mutations were determined in 39/88 (44%) patients in the study group. Q80L, S122C/N, S138W were defined as potential substitutions (6/88 patients; 7%); R109K, R117C, S122G, I132V, I170V, N174S were described as potential resistance (34/88 patients; 39%); V36L, T54S, V55A, Q80H were

Role of gemfibrozil as an inhibitor of CYP2C8 and membrane transporters.

Science.gov (United States)

Tornio, Aleksi; Neuvonen, Pertti J; Niemi, Mikko; Backman, Janne T

2017-01-01

Cytochrome P450 (CYP) 2C8 is a drug metabolizing enzyme of major importance. The lipid-lowering drug gemfibrozil has been identified as a strong inhibitor of CYP2C8 in vivo. This effect is due to mechanism-based inhibition of CYP2C8 by gemfibrozil 1-O-β-glucuronide. In vivo, gemfibrozil is a fairly selective CYP2C8 inhibitor, which lacks significant inhibitory effect on other CYP enzymes. Gemfibrozil can, however, have a smaller but clinically meaningful inhibitory effect on membrane transporters, such as organic anion transporting polypeptide 1B1 and organic anion transporter 3. Areas covered: This review describes the inhibitory effects of gemfibrozil on CYP enzymes and membrane transporters. The clinical drug interactions caused by gemfibrozil and the different mechanisms contributing to the interactions are reviewed in detail. Expert opinion: Gemfibrozil is a useful probe inhibitor of CYP2C8 in vivo, but its effect on membrane transporters has to be taken into account in study design and interpretation. Moreover, gemfibrozil could be used to boost the pharmacokinetics of CYP2C8 substrate drugs. Identification of gemfibrozil 1-O-β-glucuronide as a potent mechanism-based inhibitor of CYP2C8 has led to recognition of glucuronide metabolites as perpetrators of drug-drug interactions. Recently, also acyl glucuronide metabolites of clopidogrel and deleobuvir have been shown to strongly inhibit CYP2C8.

Synthesis and structure-activity relationship of α-keto amides as enterovirus 71 3C protease inhibitors.

Science.gov (United States)

Zeng, Debin; Ma, Yuying; Zhang, Rui; Nie, Quandeng; Cui, Zhengjie; Wang, Yaxin; Shang, Luqing; Yin, Zheng

2016-04-01

α-Keto amide derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. structure-activity relationship (SAR) study indicated that small moieties were primarily tolerated at P1' and the introduction of para-fluoro benzyl at P2 notably improved the potency of inhibitor. Inhibitors 8v, 8w and 8x exhibited satisfactory activity (IC50=1.32±0.26μM, 1.88±0.35μM and 1.52±0.31μM, respectively) and favorable CC50 values (CC50>100μM). α-Keto amide may represent a good choice as a warhead for EV71 3C(pro) inhibitor. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibitors of HIV-1 maturation: Development of structure-activity relationship for C-28 amides based on C-3 benzoic acid-modified triterpenoids.

Science.gov (United States)

Swidorski, Jacob J; Liu, Zheng; Sit, Sing-Yuen; Chen, Jie; Chen, Yan; Sin, Ny; Venables, Brian L; Parker, Dawn D; Nowicka-Sans, Beata; Terry, Brian J; Protack, Tricia; Rahematpura, Sandhya; Hanumegowda, Umesh; Jenkins, Susan; Krystal, Mark; Dicker, Ira B; Meanwell, Nicholas A; Regueiro-Ren, Alicia

2016-04-15

We have recently reported on the discovery of a C-3 benzoic acid (1) as a suitable replacement for the dimethyl succinate side chain of bevirimat (2), an HIV-1 maturation inhibitor that reached Phase II clinical trials before being discontinued. Recent SAR studies aimed at improving the antiviral properties of 2 have shown that the benzoic acid moiety conferred topographical constraint to the pharmacophore and was associated with a lower shift in potency in the presence of human serum albumin. In this manuscript, we describe efforts to improve the polymorphic coverage of the C-3 benzoic acid chemotype through modifications at the C-28 position of the triterpenoid core. The dimethylaminoethyl amides 17 and 23 delivered improved potency toward bevirimat-resistant viruses while increasing C24 in rat oral PK studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Epidemiology of Non-hereditary Angioedema

DEFF Research Database (Denmark)

Madsen, Flemming; Attermann, Jorn; Linneberg, Allan

2012-01-01

The prevalence of non-hereditary angioedema was investigated in a general population sample (n = 7,931) and in a sample of Danish patients (n = 7,433) tested for deficiency of functional complement C1 esterase inhibitor protein (functional C1 INH). The general population sample (44% response rate...

In-gel detection of esterase-like albumin activity: Characterization of esterase-free sera albumin and its putative role as non-invasive biomarker of hepatic fibrosis

Directory of Open Access Journals (Sweden)

Areeba Ahmad

2017-07-01

Full Text Available Albumin is a globular and un-glycosylated multifunctional plasma protein and thus correlated with several human diseases. Owing to esterase contamination, albumin levels are usually misleading. In this study, we propose methodical accuracy for albumin estimation taking healthy and fibrotic rats. Liver fibrosis in rats was generated by N′-Nitrosodimethylamine (NDMA (10 mg/kg body weight within three weeks followed by its confirmation through H&E and immunohistochemical staining for α-SMA expression. Animal sera were screened by native polyacrylamide gel electrophoresis (native-PAGE (7.5%. In-gel esterase-like albumin activity was detected using α- and β-naphthyl acetate (5.58 × 10−3 mM; pH 7.5 as substrate. Sera albumin was purified from unstained PA gel-slices through electroelution. Subsequent to conformation of albumin purity by its molecular weight determination using SDS–PAGE (10% and peptide mass fingerprinting by MALDI-TOF-MS, samples were treated with different concentrations of urea. Urea-treated albumins were screened for esterase activity, conformational change and, albumin levels by immunoblotting. Our results demonstrate that esterase-like albumin activity in rat sera albumin is located in domain-III. The esterase-like activity remains detectable up to 4 M urea, which diminishes with increasing urea concentrations. Further, immunoblotting of urea-treated albumin samples displays a significant decline in purified protein bands, indicating hypoalbuminemia during hepatic fibrosis in rats. In conclusion, the present approach of albumin separation and estimation is of potential interest and may be recommended for diagnostic purposes.

Biochemical and Structural Analyses of Two Cryptic Esterases in Bacteroides intestinalis and their Synergistic Activities with Cognate Xylanases.

Science.gov (United States)

Wefers, Daniel; Cavalcante, Janaina J V; Schendel, Rachel R; Deveryshetty, Jaigeeth; Wang, Kui; Wawrzak, Zdzislaw; Mackie, Roderick I; Koropatkin, Nicole M; Cann, Isaac

2017-08-04

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s -1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a Novel Esterase from Marine Environmental Genomic DNA Libraries and Its Application in Production of Free All- trans-Astaxanthin.

Science.gov (United States)

Lu, Ping; Gao, Xinwei; Dong, Hao; Liu, Zhen; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

2018-03-21

Astaxanthin is a pigment with various functions. Free astaxanthin is obtained mainly through saponification methods, which could result in many byproducts. Enzymatic methods using lipases have been used in a few cases, while there are no reports on the use of esterases for the production of free astaxanthin. Herein we present the screening and identification of a novel esterase (Est3-14) from a marine mud metagenomic library. Est3-14 is pH-sensitive and keeps good stability in alkaline buffers (residual activity 94%, pH 8.0, 4 °C, and 36 h). Meanwhile, Est3-14 keeps a good stability in the medium temperature condition (residual activity 56.7%, pH 8.0, 40 °C, and 84 h). Est3-14 displayed high hydrolysis activity to prepare free all- trans-astaxanthin in biphasic systems. Furthermore, under optimal conditions (0.5 mL ethanol, 6 mL 0.1 M Tris-HCl buffer, pH 8.0, 0.5% (w/v) H. pluvialis oil, 40 °C), the hydrolytic conversion ratio was 99.3% after 36 h.

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

Energy Technology Data Exchange (ETDEWEB)

Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning; Zhang, Dawei; Tasker, Andrew S.; Bürli, Roland; Hungate, Randall; Yu, Violeta; Nguyen, Yen; Whittington, Douglas A.; Meagher, Kristin L.; Plant, Matthew; Tudor, Yanyan; Schrag, Michael; Xu, Yang; Ng, Gordon Y.; Hu, Essa (Amgen)

2010-01-12

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

Science.gov (United States)

Hovingh, Elise S; van den Broek, Bryan; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H M; Jongerius, Ilse

2017-07-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

Science.gov (United States)

Hovingh, Elise S.; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H. M.

2017-01-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis. PMID:28742139

Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima

Energy Technology Data Exchange (ETDEWEB)

Sun, Lei [Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen (Netherlands); Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Levisson, Mark; Hendriks, Sjon; Akveld, Twan; Kengen, Servé W. M. [Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen (Netherlands); Dijkstra, Bauke W. [Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Oost, John van der, E-mail: john.vanderoost@wur.nl [Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen (Netherlands)

2007-09-01

A thermostable esterase (EstA) from Thermotoga maritima was cloned and purified. Crystals of EstA and its selenomethionine derivative were grown and diffract to beyond 2.6 Å resolution at 100 K using synchrotron radiation. A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 Å resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 Å. There are two molecules in the asymmetric unit, with a V{sub M} of 2.9 Å{sup 3} Da{sup −1} and 58% solvent content.

International consensus and practical guidelines on the gynecologic and obstetric management of female patients with hereditary angioedema caused by C1 inhibitor deficiency

DEFF Research Database (Denmark)

Caballero, Teresa; Farkas, Henriette; Bouillet, Laurence

2012-01-01

devices, and progestins can be used. Pregnancy: Attenuated androgens are contraindicated and should be discontinued before attempting conception. Plasma-derived human C1 inhibitor concentrate (pdhC1INH) is preferred for acute treatment, short-term prophylaxis, or long-term prophylaxis. Tranexamic acid...

Pathophysiological roles of aldo-keto reductases (AKR1C1 and AKR1C3) in development of cisplatin resistance in human colon cancers.

Science.gov (United States)

Matsunaga, Toshiyuki; Hojo, Aki; Yamane, Yumi; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira

2013-02-25

Cisplatin (cis-diamminedichloroplatinum, CDDP) is widely used for treatment of patients with solid tumors formed in various organs including the lung, prostate and cervix, but is much less sensitive in colon and breast cancers. One major factor implicated in the ineffectiveness has been suggested to be acquisition of the CDDP resistance. Here, we established the CDDP-resistant phenotypes of human colon HCT15 cells by continuously exposing them to incremental concentrations of the drug, and monitored expressions of aldo-keto reductases (AKRs) 1A1, 1B1, 1B10, 1C1, 1C2 and 1C3. Among the six AKRs, AKR1C1 and AKR1C3 are highly induced with the CDDP resistance. The resistance lowered the sensitivity toward cellular damages evoked by oxidative stress-derived aldehydes, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal that are detoxified by AKR1C1 and AKR1C3. Overexpression of AKR1C1 or AKR1C3 in the parental HCT15 cells mitigated the cytotoxicity of the aldehydes and CDDP. Knockdown of both AKR1C1 and AKR1C3 in the resistant cells or treatment of the cells with specific inhibitors of the AKRs increased the sensitivity to CDDP toxicity. Thus, the two AKRs participate in the mechanism underlying the CDDP resistance probably via detoxification of the aldehydes resulting from enhanced oxidative stress. The resistant cells also showed an enhancement in proteolytic activity of proteasome accompanied by overexpression of its catalytic subunits (PSMβ9 and PSMβ10). Pretreatment of the resistant cells with a potent proteasome inhibitor Z-Leu-Leu-Leu-al augmented the CDDP sensitization elicited by the AKR inhibitors. Additionally, the treatment of the cells with Z-Leu-Leu-Leu-al and the AKR inhibitors induced the expressions of the two AKRs and proteasome subunits. Collectively, these results suggest the involvement of up-regulated AKR1C1, AKR1C3 and proteasome in CDDP resistance of colon cancers and support a chemotherapeutic role for their inhibitors. Copyright © 2012 Elsevier Ireland

A novel esterase from Saccharomyces carlsbergensis, a possible function for the yeast TIP1 gene

DEFF Research Database (Denmark)

Horsted, M W; Dey, E S; Holmberg, S

1998-01-01

An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16.9 kDa. The optimal pH for activity was in the range...

Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*

Science.gov (United States)

Mautner, Henry G.; Bartels, Eva

1970-01-01

p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholine-receptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or p-nitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptor-activator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue. PMID:5272331

Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

International Nuclear Information System (INIS)

Kacham, R.; Karanth, S.; Baireddy, P.; Liu, J.; Pope, C.

2006-01-01

We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequential dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats are less

MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

DEFF Research Database (Denmark)

Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.

2015-01-01

Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors...... as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult...... zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance...

Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

International Nuclear Information System (INIS)

Herholz, Karl

2008-01-01

Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

Overexpression of esterase D in kidney from trisomy 13 fetuses

Energy Technology Data Exchange (ETDEWEB)

Loughna, S.; Moore, G. (Institute of Obstetrics and Gynaecology, London (United Kingdom)); Gau, G.; Blunt, S. (Cytogenetics Lab., London (United Kingdom)); Nicolaides, K. (King' s College School of Medicine and Dentistry, London (United Kingdom))

1993-10-01

Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. 34 refs., 3 figs., 2 tabs.

Isolation of natural inhibitors of papain obtained from Carica papaya latex

Directory of Open Access Journals (Sweden)

Rubens Monti

2004-09-01

Full Text Available Studies were carried out to natural papain inhibitor from papaya latex. Fresh latex from green fruits of Carica papaya was collected and immediately transported in ice bath to the lab, from which three fractions with inhibitor effect of esterase papain activity were isolated by latex dialysis, Sephadex G-25 gel filtration and ionic exchange chromatography in SP-Sephadex C-25. The isolated fractions, identified as inhibitors I and II, showed a negative reaction with ninhydrin; however, the fraction identified as P-III showed positive reaction with ninhydrin. Kinetics data showed non-competitive inhibition (inhibitor I and uncompetitive (inhibitors II and P-III.Este trabalho apresenta novos dados sobre inibidores naturais de papaína. O látex fresco de frutos verdes de Carica papaya foi coletado pela manhã em plantações da região de Araraquara, SP, Brasil e imediatamente transportado ao laboratório em banho de gelo. Três frações com efeito inibitório da atividade esterásica da papaína foram isoladas a partir do látex fresco, através de diálise, filtração em Sephadex G-25 e cromatografia em SP-Sephadex C-25. As frações isoladas identificadas como inibidores I e II, mostraram reação negativa à ninidrina; entretanto, a fração identificada como P-III mostrou reação positiva. Dados cinéticos revelaram inibição não-competitiva (inibidor I e incompetitiva (inibidores II e P-III.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

DEFF Research Database (Denmark)

Harholt, Jesper; Bach, Inga Christensen; Lind Bouquin, Solveig

2010-01-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used....... Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase......-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan...

Efficacy of c-Met inhibitor for advanced prostate cancer

International Nuclear Information System (INIS)

Tu, William H; Zhu, Chunfang; Clark, Curtis; Christensen, James G; Sun, Zijie

2010-01-01

Aberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR) signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC). Therefore, we directly assessed the efficacy of c-Met inhibition during androgen ablation on the growth and progression of prostate cancer. We tested two c-Met small molecule inhibitors, PHA-665752 and PF-2341066, for anti-proliferative activity by MTS assay and cell proliferation assay on human prostate cancer cell lines with different levels of androgen sensitivity. We also used renal subcapsular and castrated orthotopic xenograft mouse models to assess the effect of the inhibitors on prostate tumor formation and progression. We demonstrated a dose-dependent inhibitory effect of PHA-665752 and PF-2341066 on the proliferation of human prostate cancer cells and the phosphorylation of c-Met. The effect on cell proliferation was stronger in androgen insensitive cells. The c-Met inhibitor, PF-2341066, significantly reduced growth of prostate tumor cells in the renal subcapsular mouse model and the castrated orthotopic mouse model. The effect on cell proliferation was greater following castration. The c-Met inhibitors demonstrated anti-proliferative efficacy when combined with androgen ablation therapy for advanced prostate cancer

Esterase screening using whole cells of Brazilian soil microorganisms

Energy Technology Data Exchange (ETDEWEB)

Mantovani, Simone M.; Oliveira, Luciana G. de; Marsaioli, Anita J., E-mail: anita@iqm.unicamp.b [Universidade Estadual de Campinas (IQ/UNICAMP), SP (Brazil). Inst. de Quimica

2010-07-01

A miniaturized enzymatic assay using fluorescent probes to reveal esterase producing microorganisms was optimized and applied to screen 64 soil bacterial strains. The best results were validated using traditional non-fluorogenic assays with acetyl and propanoyl phenylethanol to confirm the miniaturized results. The most active microorganisms belong to the genus Bacillus showing esterase activity and good enantiomeric ratios for the resolution of phenylethanol derivatives (E > 30). Part of the microorganisms are kept in our laboratory in glycerol or freezedried and the best microorganisms will be deposited in the CBMAI/CPQBA/UNICAMP culture collection. (author)

The effect of fixation on esterases

DEFF Research Database (Denmark)

Kirkeby, S; Moe, D

1984-01-01

The localization of reaction product for non-specific esterase from fresh and aldehyde treated glandular tissue was examined. The electrophoretical studies showed a selective inhibition of certain isoenzymes and a change in mobility of some bands caused by aldehyde fixation. In sections a granular...

Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14

NARCIS (Netherlands)

Meijers, J. C.; Chung, D. W.

1991-01-01

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts

Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure

Science.gov (United States)

Neogi, Ujjwal; RAO, Shwetha D; BONTELL, Irene; VERHEYEN, Jens; RAO, Vasudev R; GORE, Sagar C; SONI, Neelesh; SHET, Anita; SCHÜLTER, Eugen; EKSTRAND, Maria L.; WONDWOSSEN, Amogne; KAISER, Rolf; MADHUSUDHAN, Mallur S.; PRASAD, Vinayaka R; SONNERBORG, Anders

2014-01-01

A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region which is appears in protease inhibitor (PI) failure Indian HIV-1C sequences (Odds Ratio 17.1, p<0.001) but naturally present in half of untreated Ethiopian sequences. The insertion will probably restore the ALIX mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of such insertion need to be evaluated in HIV-1C dominating regions were PI-drugs are being scaled up as second line treatment options. PMID:25102091

Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

Directory of Open Access Journals (Sweden)

Tyagi Sadhna

2009-06-01

Full Text Available Abstract Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand, although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

Science.gov (United States)

Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

2015-07-01

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Epidemiology of Non-hereditary Angioedema

DEFF Research Database (Denmark)

Madsen, Flemming; Attermann, Jørn; Linneberg, Allan

2012-01-01

The prevalence of non-hereditary angioedema was investigated in a general population sample (n¿=¿7,931) and in a sample of Danish patients (n¿=¿7,433) tested for deficiency of functional complement C1 esterase inhibitor protein (functional C1 INH). The general population sample (44% response rate...

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

Science.gov (United States)

Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

2017-04-11

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Lipase and esterase: to what extent can this classification be applied accurately?

Directory of Open Access Journals (Sweden)

Danielle Branta Lopes

2011-09-01

Full Text Available Enzyme technology is an ever-growing field of knowledge and, in recent years, this technology has raised renewed interest, due to the search for new paradigms in several productive processes. Lipases, esterases and cutinases are enzymes used in a wide range of processes involving synthesis and hydrolysis reactions. The objective of this work was to investigate and compare the specific lipase and esterase activities of five enzymes - four already classified as lipases and one classified as cutinase - in the presence of natural and synthetic substrates. All tested enzymes presented both esterase and lipase specific activities. The highest specific esterase activity was observed for Aspergillus 1068 lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate, while the highest specific lipase activity was observed for Geotrichum sp. lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate. These results display some interface-independent lipolytic activity for all lipases tested. This is in accordance with the rationale that a new and broader definition of lipases may be necessary.

Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

Directory of Open Access Journals (Sweden)

Kazi Abdus Salam

2013-01-01

Full Text Available Currently, hepatitis C virus (HCV infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin. The new therapy has significantly improved sustained virologic response (SVR; however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.

Inhibitors of the bacterial cell wall biosynthesis enzyme MurC.

Science.gov (United States)

Reck, F; Marmor, S; Fisher, S; Wuonola, M A

2001-06-04

A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

Science.gov (United States)

Komiya, Dai; Hori, Akane; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Koseki, Takuya; Fushinobu, Shinya

2017-10-15

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis ( Al AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. Al AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that Al AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of A lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of

Zymography Detection of a Bacterial Extracellular Thermoalkaline Esterase/Lipase Activity.

Science.gov (United States)

Tapizquent, María; Fernández, Marleny; Barreto, Georgina; Hernández, Zully; Contreras, Lellys M; Kurz, Liliana; Wilkesman, Jeff

2017-01-01

Lipases are esterases that occur widely in nature, yet those with commercial relevance are exclusively from microbial origin. Glycerol and long-chain fatty acids are the products after hydrolysis of esters bonds in saponifiable lipids catalyzed by lipases. In this work, we describe lipase/esterase activity contained in cell-free fractions from thermophilic bacteria, cultured in medium containing olive oil. Analysis of the cell-free fractions by electrotransference zymography, using tributyrin as substrate, revealed bands corresponding to lipase activity. The method is simple, fast, and inexpensive.

Alicyclobacillus acidocaldarius Thermophilic Esterase EST2's Activity in Milk and Cheese Models

NARCIS (Netherlands)

Mandrich, L.; Manco, M.; Rossie, M.; Floris, E.; Jansen-van den Bosch, T.; Smit, G.; Wouters, J.A.

2006-01-01

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase.

Science.gov (United States)

Rowley, David C; Hansen, Mark S T; Rhodes, Denise; Sotriffer, Christoph A; Ni, Haihong; McCammon, J Andrew; Bushman, Frederic D; Fenical, William

2002-11-01

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

Esterase resistant to inactivation by heavy metals

KAUST Repository

El, Dorry Hamza; Siam, Rania; Mohamed, Yasmine M.

2014-01-01

EstATII is an esterase that a halotolerant, thermophilic and resistant to a spectrum of heavy metals including toxic concentration of metals. It was isolated from the lowest convective layer of the Atlantis II Red Sea brine pool. The Atlantis II

Effects of phosphodiesterase inhibitors on atrial dynamics induced by C-type natriuretic peptide in isolated beating rabbit atria

International Nuclear Information System (INIS)

Ding Dazhi; Cui Xun; Jin Xiunan; Lan Ying; Liu Liping; Hong Lan

2010-01-01

Objective: To investigate the effects of phosphodiesterase inhibitors (PDEI) on atrial dynamics induced by C-type natriuretic peptide (CNP) and the contents of cyclic nucleotide (cAMP, cGMP) in isolated beating rabbit atria. Methods: After the rabbits had been anesthetized, the hearts were removed rapidly. The left auricles were isolated and fixed on the atrial perfusion system. The atrial stroke volume and the pulse pressure were observed by CNP with or without PDEIs pretreatment. The contents of cAMP and cGMP were measured by radioimmunoassay. Results: (1)Compared with control cycle group, CNP (30.0 nmol · L -1 ) obviously decreased the atrial stroke volume and pulse pressure (P 0.05). (2)Compared with control cycle group, IBMX(1000.0 nmol · L -1 ), a non-selective inhibitor of PDE, significantly increased the atrial stroke volume, pulse pressure, cAMP and cGMP contents (P -1 ) plus CNP (30.0 nmol · L -1 )group and IBMX group (P>0.05). (3)Compared with control cycle group, EHNA(30.0 nmol · L -1 ), an inhibitor of PDE2, obviously decreased the atrial stroke volume and pulse pressure (P 0.05). EHNA(30.0 nmol · L -1 ) plus CNP (30.0 nmol · L -1 ) showed similar roles with EHNA only. (4)Compared with control cycle group, milrinone (1.0 nmol · L -1 ), an inhibitor of PDE3, significantly increased the content of cAMP (P 0.05). CNP (30.0 nmol · L -1 ) obviously decreased the atrial stroke volume and pulse pressure (P 0.05). Conclusion: CNP can inhibit atrial dynamics by increasing the content of cGMP, the different inhibitors of PDEs play different roles in the CNP-induced inhibition of atrial dynamics in isolated beating rabbit atria. (authors)

A nomogram to estimate the HbA1c response to different DPP-4 inhibitors in type 2 diabetes: a systematic review and meta-analysis of 98 trials with 24 163 patients

Science.gov (United States)

Esposito, Katherine; Chiodini, Paolo; Maiorino, Maria Ida; Capuano, Annalisa; Cozzolino, Domenico; Petrizzo, Michela; Bellastella, Giuseppe; Giugliano, Dario

2015-01-01

Objectives To develop a nomogram for estimating the glycated haemoglobin (HbA1c) response to different dipeptidyl peptidase-4 (DPP-4) inhibitors in type 2 diabetes. Design A systematic review and meta-analysis of randomised controlled trials (RCTs) of DPP-4 inhibitors (vildagliptin, sitagliptin, saxagliptin, linagliptin and alogliptin) on HbA1c were conducted. Electronic searches were carried out up to December 2013. Trials were included if they were carried out on participants with type 2 diabetes, lasted at least 12 weeks, included at least 30 participants and had a final assessment of HbA1c. A random effect model was used to pool data. A nomogram was used to represent results of the metaregression model. Participants Adults with type 2 diabetes. Interventions Any DPP-4 inhibitor (vildagliptin, sitagliptin, saxagliptin, linagliptin or alogliptin). Outcome measures The HbA1c response to each DPP-4 inhibitor within 1 year of therapy. Results We screened 928 citations and reviewed 98 articles reporting 98 RCTs with 100 arms in 24 163 participants. There were 26 arms with vildagliptin, 37 with sitagliptin, 13 with saxagliptin, 13 with linagliptin and 11 with alogliptin. For all 100 arms, the mean baseline HbA1c value was 8.05% (64 mmol/mol); the decrease of HbA1c from baseline was −0.77% (95% CI −0.82 to −0.72%), with high heterogeneity (I2=96%). Multivariable metaregression model that included baseline HbA1c, type of DPP-4 inhibitor and fasting glucose explained 58% of variance between studies, with no significant interaction between them. Other factors, including age, previous diabetes drugs and duration of treatment added low predictive power (HbA1c reduction from baseline using the type of DPP-4 inhibitor, baseline values of HbA1c and fasting glucose. Conclusions Baseline HbA1c level and fasting glucose explain most of the variance in HbA1c change in response to DPP-4 inhibitors: each increase of 1.0% units HbA1c provides a 0.4–0.5% units greater

Immobilization of cholesterol esterase and cholesterol oxidase onto sol-gel films for application to cholesterol biosensor

International Nuclear Information System (INIS)

Singh, Suman; Singhal, Rahul; Malhotra, B.D.

2007-01-01

Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto tetraethylorthosilicate (TEOS) sol-gel films. The tetraethylorthosilicate sol-gel/ChEt/ChOx enzyme films thus prepared have been characterized using scanning electron microscopic (SEM), UV-vis spectroscopic, Fourier-transform-infrared (FTIR) spectroscopic and amperometric techniques, respectively. The results of photometric measurements carried out on tetraethylorthosilicate sol-gel/ChEt/ChOx reveal thermal stability up to 55 deg. C, response time as 180 s, linearity up to 780 mg dL -1 (12 mM), shelf life of 1 month, detection limit of 12 mg dL -1 and sensitivity as 5.4 x 10 -5 Abs. mg -1 dL -1

Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors

DEFF Research Database (Denmark)

Li, Yi-Ping; Ramirez, Santseharay; Humes, Daryl

2014-01-01

BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A...... daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all...... protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency...

Pectin Methyl Esterase Activity Change in Intermediate Moisture Sun-Dried Figs after Storage

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Dilek Demirbüker Kavak

2015-12-01

Full Text Available Intermediate moisture fruits can be obtained by rehydrating dried fruits. Intermediate moisture fruits are suitable for direct consumption compared to dry fruits and can be directly used in the production of various products such as bakery products, dairy products and candies. Aim of this study is to compare the pectin methyl esterase (PME activity of intermediate moisture figs which causes softening of the texture and to compare their microbial stability after 3 months storage period. For this purpose, dried figs were rehydrated in 30 and 80° C water until they reach 30% moisture content. Rehydrated samples were stored for 3 months at +4°C. Results showed that there was no statistically significant difference between the control samples and the samples rehydrated at 80°C according to the total viable counts. At the end of the storage period, results of residual PME activity in control samples was 24.1 μmol COOH min-1g-1, while it was found 17.4 μmol COOH min-1g-1 in samples rehydrated at 80°C. As a result rehydration conducted at 80°C provided 28% reduction in PME activity compared to the control samples rehydrated at 30°C, although it did not affect the microbial load significantly after storage.

Design and optimization of a series of 1-sulfonylpyrazolo[4,3-b]pyridines as selective c-Met inhibitors.

Science.gov (United States)

Ma, Yuchi; Sun, Guangqiang; Chen, Danqi; Peng, Xia; Chen, Yue-Lei; Su, Yi; Ji, Yinchun; Liang, Jin; Wang, Xin; Chen, Lin; Ding, Jian; Xiong, Bing; Ai, Jing; Geng, Meiyu; Shen, Jingkang

2015-03-12

c-Met has emerged as an attractive target for targeted cancer therapy because of its abnormal activation in many cancer cells. To identify high potent and selective c-Met inhibitors, we started with profiling the potency and in vitro metabolic stability of a reported hit 7. By rational design, a novel sulfonylpyrazolo[4,3-b]pyridine 9 with improved DMPK properties was discovered. Further elaboration of π-π stacking interactions and solvent accessible polar moieties led to a series of highly potent and selective type I c-Met inhibitors. On the basis of in vitro and in vivo pharmacological and pharmacokinetics studies, compound 46 was selected as a preclinical candidate for further anticancer drug development.

Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system

International Nuclear Information System (INIS)

Mangas, Iris; Vilanova, Eugenio; Estévez, Jorge

2011-01-01

Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and “ongoing inhibition”) of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and the model applied and may be considered an external validation. The most sensitive component (Eα) for paraoxon (11–23% of activity, I 50 (30 min) = 9–11 nM) is also the most sensitive for mipafox (I 50 (30 min) = 4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (Eβ, 71–84% of activity; I 50 (30 min) = 1216 nM) is practically resistant to mipafox. The third component (Eγ, 5–8% of activity) is paraoxon resistant and has I 50 (30 min) of 3.4 μM with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies. -- Research Highlights: ► Paraoxon and mipafox interactions have been evaluated with chicken soluble brain esterases. ► The paraoxon inhibition was analyzed considering the simultaneous spontaneous reactivation. ► The best-fitting kinetic models were compatible with a three enzymatic components. ► The amplitudes of the

Dampak Hipoksia Sistemik terhadap Malondialdehida, Glial Fibrillary Acidic Protein dan Aktivitas Asetilkolin Esterase Otak Tikus

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Andriani Andriani

2016-09-01

Full Text Available Hipoksia sistemik menyebabkan berkurangnya oksigen dan energi di otak sehingga memicupenglepasan neurotransmiter asetilkolin, meningkatkan radikal bebas dan glial fibrillary acidic protein (GFAPyang berfungsi menjaga kekuatan membran. Tujuan penelitian untuk melihat gambaran adaptasi otak padahipoksia sistemik terhadap fungsi asetilkolin esterase, kerusakan membran sel neuron dan astrosit. Penelitiandilakukan di Laboratorium Biokimia & Biologi Molekuler FK Universitas Indonesia, pada tahun 2013.Penelitian ekperimental ini menggunakan hewan coba tikus spraque dawley yang diinduksi hipoksia sistemikyang diambil jaringan otak bagian korteks dan plasma tikus. Kelompok tikus terdiri atas kelompok kontrol,kelompok perlakuan induksi hipoksia hari ke-1, 3 hari, 5 hari dan hari ke-7. Parameter yang diukur adalahkadar malondialdehida (MDA otak dan plasma, aktivitas spesifik enzim AChE jaringan otak serta kadar GFAPjaringan otak. Hasil menunjukkan bahwa hipoksia sistemik tidak meningkatkankadar MDA otak dan plasma.Induksi hipoksia sistemik meningkatkan aktivitas spesifik enzim AChE dan kadar GFAP jaringan otak secarabermakna. Pada plasma tidak terjadi peningkatan kadar GFAP. Hipoksia sistemik selama hari ke-7 belummenyebabkan kerusakan oksidatif, namun memperlihatkan peningkatan aktivitas AChe dan adaptasi astrositmelalui peningkatan GFAP. Kata kunci: hipoksia, astrosit, glial fibrillary acidic protein, malondialdehida, asetilkolin esterase   Systemic Hypoxia Effect on Rat Brain Malondialdehyde, Glial FibrillaryAcidic Protein, and Acetylcholine Esterase Activity Abstract Sistemic hypoxia causes lack of oxygen and energy in brain that trigger the release of acetylcholine,free radical and Glial fibrillary acidic protein (GFAP, a specific protein in astrocyte cells that act to strenghtenastrocite membrane. The aim of the research was to evaluate the damages of brain in systemic hypoxiathrough activity of acetylcholine esterase, neuron and astrocyte membran

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

Science.gov (United States)

Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

2015-01-01

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

Acidic-alkaline ferulic acid esterase from Chaetomium thermophilum var. dissitum: Molecular cloning and characterization of recombinant enzyme expressed in Pichia pastoris

DEFF Research Database (Denmark)

Dotsenko, Gleb; Tong, Xiaoxue; Pilgaard, Bo

2016-01-01

A novel ferulic acid esterase encoding gene CtFae, was successfully cloned from a highly esterase active strain of the thermophile ascomycetous fungus Chaetomium thermophilum var. dissitum; the gene was heterologously expressed in Pichia pastoris KM71H. The recombinant enzyme (CtFae) was purified...... to homogeneity and subsequently characterized. CtFae was active towards synthetic esters of ferulic, p-coumaric, and caffeic acids, as well as towards wide range of p-nitrophenyl substrates. Its temperature and pH optima were 55 °C and pH 6.0, respectively. Enzyme rare features were broad pH optimum, high...

Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

Energy Technology Data Exchange (ETDEWEB)

Hamers, T.; Brink, P.J. van den; Mos, L.; Linden, S.C. van der; Legler, J.; Koeman, J.H.; Murk, A.J

2003-05-01

Estrogenic potency of rainwater correlated well with organochlorine concentrations, but could not be attributed to specific pesticides. - In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between SPRING and SUMMER were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in HORT than in BACK and BULB. Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for

Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

International Nuclear Information System (INIS)

Hamers, T.; Brink, P.J. van den; Mos, L.; Linden, S.C. van der; Legler, J.; Koeman, J.H.; Murk, A.J.

2003-01-01

Estrogenic potency of rainwater correlated well with organochlorine concentrations, but could not be attributed to specific pesticides. - In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between SPRING and SUMMER were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in HORT than in BACK and BULB. Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for

1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

International Nuclear Information System (INIS)

Asare, Nana; Landvik, Nina E.; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Lag, Marit; Holme, Jorn A.

2008-01-01

Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent

[Memantine as add-on medication to acetylcholinesterase inhibitor therapy for Alzheimer dementia].

Science.gov (United States)

Haussmann, R; Donix, M

2017-01-01

Currently available data indicate superior therapeutic effects of combination treatment for Alzheimer dementia with memantine and acetylcholine esterase inhibitors in certain clinical contexts. Out of five randomized, placebo-controlled, double-blind trials two showed superior therapeutic effects in comparison to monotherapy with acetylcholinesterase inhibitors regarding various domains. Recently published meta-analyses and cost-benefit analyses also showed positive results. Recently published German guidelines for dementia treatment also take these new data into account and recommend combination treatment in patients with severe dementia on stable donepezil medication. This article gives an overview of current evidence for combination therapy.

Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

African Journals Online (AJOL)

Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

Synthesis of[11C]LY186126, an inhibitor of phosphodiesterase

International Nuclear Information System (INIS)

Prenant, C.; Crouzel, C.; Comar, D.; Robertson, D.W.

1992-01-01

LY186126 [1,3-dihydro-1,3,3-trimethyl-5-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyriddazinyl)-2H-indol-2-one ], an analogue of the cardiotonic agent indolidan, is a potent, selective and competitive inhibitor of an isozymic form of cyclic AMP phosphodiesterase. LY186126 was labelled with carbon-11 to permit pharmacological studies in the dog myocardium by positron emission tomography. Alkylation with [ 11 C]methyl iodide of N-norLY186126 (LY-197055) allowed the production of 1.7 GBq (50mCi) of [ 11 C]LY-186126 in 40 min. The product, was purified by HPLC. (author)

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

Science.gov (United States)

Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Effect of phenobarbital on inducing insecticide tolerance and esterase changes in Aedes aegypti (Diptera: Culicidae

Directory of Open Access Journals (Sweden)

Rita de Cássia Sousa-Polezzi

2004-01-01

Full Text Available The effect of phenobarbital (PB on the induction of tolerance to the organophosphorous insecticide temephos (TE was investigated in Aedes aegypti L4 larvae submitted to two different PB-treatments:(1 continuous treatment from the egg to the larval L4 stage and (2 discontinuous treatment in which L4 larvae were exposed for 30 h. Mosquitoes from two Brazilian cities were studied: São José do Rio Preto (SJ in São Paulo State and Goiânia (GO in Goiás State. According to criterions established by World Health Organization (WHO mosquitoes from SJ are organophosphate-susceptible while mosquitoes from GO are organophosphate-resistant. For both SJ and GO larvae the two different PB-treatments resulted in significantly increased tolerance (measured by reduced mortality to 0.01mg/L TE while for larvae exposed to 0.02 mg/L TE only continuous PB-treatment resulted in significantly increased TE-tolerance. The reduction of mortality rate was greater in SJ larvae than in GO larvae, confirming data from other organisms indicating that the effect of PB is more pronounced in susceptible strains. To test if oxidase enzymes were involved in PB-induced tolerance we treated PB-pretreated SJ and GO larvae with the oxidase inhibitor piperonyl butoxide (PBO before exposure to TE and observed increased (rather than decreased tolerance, suggesting that oxidases are not involved in the tolerance process and that PB and PBO can act in concert or synergistically. Esterase patterns of PB-pretreated larvae indicated that the cholinesterases EST-13 and EST-14 are involved in the PB-induced TE- tolerance, reinforcing a previous study carried out in our laboratory which suggested that increased esterase synthesis is the mechanism responsible for the development of insecticide resistance in Aedes aegypti.

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

Science.gov (United States)

Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

2015-03-02

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Esterases no exame da estrutura populacional de Camu-camu (Myrciaria dubia (Kunth McVaugh-Myrtaceae Esterases for examining the population structure of Camu-camu (Myrciaria dubia (Kunth McVaugh-Myrtaceae

Directory of Open Access Journals (Sweden)

Aylton Saturnino Teixeira

2004-01-01

Full Text Available Dois sistemas enzimáticos (esterase e esterase-D, analisados pela técnica de eletroforese em gel de amido, em folhas jovens de plantas cultivadas em terra firme, de sementes provenientes de três amostras de populações naturais de camu-camu, Myrciaria dubia (Kunth McVaugh-Myrtaceae, procedentes de Iquitos, Boa Vista e Uatumã, revelaram a presença de 6 locos: Est-1, Est-2, Est-3, Est-4, Est-D1 e Est-D2. Dois dos seis locos gênicos examinados no presente estudo (Est-3 e Est-D2 mostraram-se polimórficos, sendo desse modo considerados valiosos no estudo de caracterização da estrutura populacional da espécie. Os padrões de polimorfismo revelados nos locos Est-3 e Est-D2 de camu-camu, são típicos de enzimas monoméricas e diméricas, respectivamente. O loco Est-3 apresentou um grande desbalanço genético dentro e entre as amostras populacionais examinadas, devido ao excessivo número observado de plantas heterozigóticas em relação ao número esperado. O loco Est-D2 apresentou um polimorfismo exclusivo para os alelos Est-D2¹,Est-D2² e Est-D2³, e um bom balanço genético na amostra populacional de Uatumã. Em função disso, dentre os demais locos gênicos aqui investigados, o loco Est-D2 parece ser o mais adequado para identificação e delimitação de prováveis estoques de camu-camu. Portanto, recomenda-se que esse loco esteja presente na lista dos marcadores isoenzimáticos a serem usados em futuras prospecções sobre genética populacional dessa espécie na região amazônica. Dados sobre a distribuição das freqüências alélicas, estimativas das distâncias genéticas, e estimativas de variação genética nos 6 locos de esterases examinados, foram eficazes na demonstração de diferenças genéticas entre as amostras populacionais examinadas da espécie. Os maiores valores de heterozigozidade média (0,1353; proporção de locos polimórficos (0,33 e número médio de alelos por loco (1,33 revelados na amostra

Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

Energy Technology Data Exchange (ETDEWEB)

Herholz, Karl [University of Manchester, Wolfson Molecular Imaging Centre, Clinical Neuroscience, Manchester (United Kingdom); University of Cologne, Cologne (Germany)

2008-03-15

Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

DEFF Research Database (Denmark)

Udatha, D.B.R.K. Gupta; Madsen, Karina Marie; Panagiotou, Gianni

2015-01-01

The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus...... sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis....... To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases....

Novel recombinant ethyl ferulate esterase from Burkholderia multivorans

CSIR Research Space (South Africa)

Rashamuse, KJ

2007-11-01

Full Text Available Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment...

Notes on electropherograms of eye-lens, muscle proteins and zymograms of muscle esterases of fish collected during the first Brazilian expedition to the Antarctica

Directory of Open Access Journals (Sweden)

Van Ngan Phan

1985-01-01

Full Text Available A preliminary study was carried out on electropherograms of eye-lens, muscle proteins and zymograms of muscle esterases of ten Notothenia larseni, six Notothenia nudifrons and one lanternfish, Electrona antarctica. The fish were collected by the R/V "Prof. W. Besnard" of the Institute of Oceanography, University of São Paulo, during the First Brazilian Expedition to Antarctica. Eye-lens proteins were analysed on cellulose acetate membrane, muscle proteins and esterases on gel of polyaorylamide. Eye-lens proteins showed three types of electropherograms for N. larseni, and two types for N. nudifrons. One of the electropherograms of N. larseni can be readily distinguished from those of N. nudifrons. Electropherograms of muscle proteins of N. larseni and N. nudifrons are very similar and, consist of sixteen to seventeen fractions. Electropherograms of muscle proteins of N. larseni are severely affected by the conservation of the extracts overnight under -20ºC. All N. nudifrons were of the same zymograms of esterases while those of N. larseni varied. Electropherograms of eye-lens and muscle proteins as well as zymograms of esterases of the lanternfish are different from those of nototheniids.Foi realizado um estudo preliminar sobre eletroferogramas de proteínas de cristalino e de músculo esquelético, e zimogramas de esterases de músculo esquelético de dez Notothenia larseni, seis Notothenia nudifrons e de um peixe-lanterna, Electrona antarctica. Os peixes foram coletados pelo N/Oc. "Prof. W. Besnard" do Instituto Oceanográfico da Universidade de São Paulo durante a I Expedição Brasileira à Antártica. As proteinas do cristalino foram analisadas em membranas de acetato de celulose, enquanto que as proteínas e esterases do músculo esquelético, em gel de poliacrilamida. As proteínas do cristalino apresentam três tipos distintos de eletroferogramas para N. larseni, e dois para N. nudifrons. Um dos eletroferogramas de N. larseni, pode ser

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

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Marlien Pieters

Full Text Available Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g, platelet-containing (352 g and platelet-rich plasma (200 g were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation. Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly

Some aromatic hydrazone derivatives as inhibitors for the corrosion of C-steel in phosphoric acid solution.

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Fouda, Abd El-Aziz S; Al-Sarawy, Ahmed A; Radwan, Mohamed S

2006-01-01

The effect of furfural benzoylhydrazone and its derivatives (I-VII) as corrosion inhibitors for C-steel in 1M phosphoric acid solution has been studied by weight-loss and galvanostatic polarization techniques. A significant decrease in the corrosion rate of C-steel was observed in the presence of the investigated inhibitors. This study revealed that, the inhibition efficiency increases with increasing the inhibitor concentration, and the addition of iodide ions enhances it to a considerable extent. The effect of temperature on the inhibition efficiency of these compounds was studied using weight-loss method. Activation energy (E(a)*) and other thermodynamic parameters for the corrosion process were calculated and discussed. The galvanostatic polarization data indicated that, the inhibitors were of mixed-type, but the cathode is more polarized than the anode. The adsorption of these compounds on C-steel surface has been found to obey Frumkin's adsorption isotherm. The mechanism of inhibition was discussed in the light of the chemical structure of the undertaken inhibitors.

Immobilization of cholesterol esterase and cholesterol oxidase onto sol-gel films for application to cholesterol biosensor

Energy Technology Data Exchange (ETDEWEB)

Singh, Suman [Central Mechanical Engineering Research Institute, G. Avenue, Durgapur 713209, West Bengal (India); Singhal, Rahul [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012 (India); Malhotra, B.D. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012 (India)]. E-mail: bansi.malhotra@gmail.com

2007-01-23

Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto tetraethylorthosilicate (TEOS) sol-gel films. The tetraethylorthosilicate sol-gel/ChEt/ChOx enzyme films thus prepared have been characterized using scanning electron microscopic (SEM), UV-vis spectroscopic, Fourier-transform-infrared (FTIR) spectroscopic and amperometric techniques, respectively. The results of photometric measurements carried out on tetraethylorthosilicate sol-gel/ChEt/ChOx reveal thermal stability up to 55 deg. C, response time as 180 s, linearity up to 780 mg dL{sup -1} (12 mM), shelf life of 1 month, detection limit of 12 mg dL{sup -1} and sensitivity as 5.4 x 10{sup -5} Abs. mg{sup -1} dL{sup -1}.

Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides.

Science.gov (United States)

Wang, Zhiling; Zhao, Zhong; Cheng, Xiaofei; Liu, Suqi; Wei, Qin; Scott, Ian M

2016-02-01

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

The monotony of transferrin and esterase electrophoretic patterns in pirarucu, Arapaima gigas (Schinz, 1822) from Santa Cruz Lake, Tefé River, Amazonas, Brazil.

Science.gov (United States)

Teixeira, A S

2008-05-07

Starch gel electrophoresis was used for examining the transferrin gene locus (Tf) and two esterase gene loci (Est-1 and Est-D1) of a pirarucu (Arapaima gigas) population sample collected from Santa Cruz Lake, Tefé River, Amazonas, Brazil. The Tf locus was tentatively classified as being polymorphic, showing two double-banded patterns (Tf(12) and Tf(22)) of the three theoretically expected ones (Tf(11), Tf(12) and Tf(22)), presumably controlled by two co-dominant alleles, Tf(1) and Tf(2). The monotony detected in pirarucu Tf locus genotypes showing a very high proportion of the double-banded heterozygote pattern Tf(12) (95% of the sampled individuals) may indicate the possibility of their having come from representatives of the same brood begotten by a pair of fish, where a single-banded Tf(11) homozygote pattern male would have crossed with a single-banded Tf(22) homozygote pattern female, or vice versa. One zone of electrophoretic activity was detected in esterase, presumably controlled by a monomorphic Est-1 locus with the fixed allele Est-1(1) where all individuals showed the single-banded Est-1(11) homozygote pattern. Esterase-D also displayed one zone of electrophoretic activity, presumably controlled by a monomorphic Est-D1 locus with a fixed allele Est-D1(1) where all individuals revealed the single-banded Est-D1(11) genotype pattern. The monotony comprised by single-banded genotype patterns in both esterase systems tested may also indicate the possibility of the individuals from the sample examined having come from representatives of the same brood begotten by a pair of fish with both the male and female having the same genotypes.

Identification and characterization of a GDSL esterase gene located proximal to the swr quorum-sensing system of Serratia liquefaciens MG1

DEFF Research Database (Denmark)

Riedel, K.; Talker-Huiber, D.; Givskov, Michael Christian

2003-01-01

direction, designated estA, which encodes an esterase that belongs to family II of lipolytic enzymes. EstA was heterologously expressed in Escherichia coli, and the substrate specificity of the enzyme was determined in crude extracts. With the aid of zymograms visualizing EstA on polyacrylamide gels...... and by the analysis of a transcriptional fusion of the estA promoter to the promoterless lux4B genes, we showed that expression of the esterase is not regulated by the swr quorum-sensing system. An estA mutant was generated and was found to exhibit growth defects on minimal medium containing Tween 20 or Tween 80...

The proton pump inhibitor, omeprazole, but not lansoprazole or pantoprazole, is a metabolism-dependent inhibitor of CYP2C19: implications for coadministration with clopidogrel.

Science.gov (United States)

Ogilvie, Brian W; Yerino, Phyllis; Kazmi, Faraz; Buckley, David B; Rostami-Hodjegan, Amin; Paris, Brandy L; Toren, Paul; Parkinson, Andrew

2011-11-01

As a direct-acting inhibitor of CYP2C19 in vitro, lansoprazole is more potent than omeprazole and other proton pump inhibitors (PPIs), but lansoprazole does not cause clinically significant inhibition of CYP2C19 whereas omeprazole does. To investigate this apparent paradox, we evaluated omeprazole, esomeprazole, R-omeprazole, lansoprazole, and pantoprazole for their ability to function as direct-acting and metabolism-dependent inhibitors (MDIs) of CYP2C19 in pooled human liver microsomes (HLM) as well as in cryopreserved hepatocytes and recombinant CYP2C19. In HLM, all PPIs were found to be direct-acting inhibitors of CYP2C19 with IC(50) values varying from 1.2 μM [lansoprazole; maximum plasma concentration (C(max)) = 2.2 μM] to 93 μM (pantoprazole; C(max) = 6.5 μM). In addition, we identified omeprazole, esomeprazole, R-omeprazole, and omeprazole sulfone as MDIs of CYP2C19 (they caused IC(50) shifts after a 30-min preincubation with NADPH-fortified HLM of 4.2-, 10-, 2.5-, and 3.2-fold, respectively), whereas lansoprazole and pantoprazole were not MDIs (IC(50) shifts lansoprazole, or pantoprazole, as irreversible (or quasi-irreversible) MDIs of CYP2C19. These results have important implications for the mechanism of the clinical interaction reported between omeprazole and clopidogrel, as well as other CYP2C19 substrates.

Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site.

Science.gov (United States)

Du, Yongli; Zhang, Yanhui; Ling, Hao; Li, Qunyi; Shen, Jingkang

2018-01-20

PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great selectivity over the highly homologous TCPTP and other PTPs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

Directory of Open Access Journals (Sweden)

Pablo Luis Santo-Orihuela

2013-12-01

Full Text Available The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia and from domiciliary areas, including El Palmar (Bolivia and La Pista (Argentina. Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR value (44.90 compared to the susceptible reference strain (NFS, whereas the Veinte de Octubre samples had the lowest value (0.50. Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively. The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP.

Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

Energy Technology Data Exchange (ETDEWEB)

Rong, Libin [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory

2009-01-01

Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

Usefulness of Leukocyte Esterase Test Versus Rapid Strep Test for Diagnosis of Acute Strep Pharyngitis

Directory of Open Access Journals (Sweden)

Kumara V. Nibhanipudi MD

2015-08-01

Full Text Available Objective: A study to compare the usage of throat swab testing for leukocyte esterase on a test strip(urine dip stick-multi stick to rapid strep test for rapid diagnosis of Group A Beta hemolytic streptococci in cases of acute pharyngitis in children. Hypothesis: The testing of throat swab for leukocyte esterase on test strip currently used for urine testing may be used to detect throat infection and might be as useful as rapid strep. Methods: All patients who come with a complaint of sore throat and fever were examined clinically for erythema of pharynx, tonsils and also for any exudates. Informed consent was obtained from the parents and assent from the subjects. 3 swabs were taken from pharyngo-tonsillar region, testing for culture, rapid strep & Leukocyte Esterase. Results: Total number is 100. Cultures 9(+; for rapid strep== 84(- and16 (+; For LE== 80(- and 20(+ Statistics: From data configuration Rapid Strep versus LE test don’t seem to be a random (independent assignment but extremely aligned. The Statistical results show rapid and LE show very agreeable results. Calculated Value of Chi Squared Exceeds Tabulated under 1 Degree Of Freedom (P<.0.0001 reject Null Hypothesis and Conclude Alternative Conclusions: Leukocyte esterase on throat swab is as useful as rapid strep test for rapid diagnosis of strep pharyngitis on test strip currently used for urine dip stick causing acute pharyngitis in children.

Subnanomolar Inhibitor of Cytochrome bc1 Complex Designed via Optimizing Interaction with Conformationally Flexible Residues

Science.gov (United States)

Zhao, Pei-Liang; Wang, Le; Zhu, Xiao-Lei; Huang, Xiaoqin; Zhan, Chang-Guo; Wu, Jia-Wei; Yang, Guang-Fu

2009-01-01

Cytochrome bc1 complex (EC 1.10.2.2, bc1), an essential component of the cellular respiratory chain and the photosynthetic apparatus in photosynthetic bacteria, has been identified as a promising target for new drugs and agricultural fungicides. X-ray diffraction structures of the free bc1 complex and its complexes with various inhibitors revealed that the phenyl group of Phe274 in the binding pocket exhibited significant conformational flexibility upon different inhibitors binding to optimize respective π-π interactions, whereas the side chains of other hydrophobic residues showed conformational stability. Therefore, in the present study, a strategy of optimizing the π-π interaction with conformationally flexible residues was proposed to design and discover new bc1 inhibitors with a higher potency. Eight new compounds were designed and synthesized, among which compound 5c with a Ki value of 570 pM was identified as the most promising drug or fungicide candidate, significantly more potent than the commercially available bc1 inhibitors including azoxystrobin (AZ), kresoxim-methyl (KM), and pyraclostrobin (PY). To our knowledge, this is the first bc1 inhibitor discovered from structure-based design with a potency of subnanomolar Ki value. For all of the compounds synthesized and assayed, the calculated binding free energies correlated reasonably well with the binding free energies derived from the experimental Ki values with a correlation coefficient of r2 = 0.89. The further inhibitory kinetics studies revealed that compound 5c is a non-competitive inhibitor with respect to substrate cytochrome c, but is a competitive inhibitor with respect to substrate ubiquinol. Due to its subnanomolar Ki potency and slow dissociation rate constant (k−0 = 0.00358 s−1), compound 5c could be used as a specific probe for further elucidation of the mechanism of bc1 function and as a new lead compound for future drug discovery. PMID:19928849

The potential of P1 site alterations in peptidomimetic protease inhibitors as suggested by virtual screening and explored by the use of C-C-coupling reagents.

Science.gov (United States)

Weik, Steffen; Luksch, Torsten; Evers, Andreas; Böttcher, Jark; Sotriffer, Christoph A; Hasilik, Andrej; Löffler, Hans-Gerhard; Klebe, Gerhard; Rademann, Jörg

2006-04-01

A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer-supported C-acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C-acylation can be conducted without racemization. Diastereoselective reduction was effected with L-selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with approximately 500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4-bromophenyl, and 4-nitrophenyl enhanced the inhibitory activity (IC50) by up to 70-fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9-fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.

Cholesterol esterase inhibitory activity of bioactives from leaves of Mangifera indica L

Science.gov (United States)

Gururaja, G. M.; Mundkinajeddu, Deepak; Dethe, Shekhar M.; Sangli, Gopala K.; Abhilash, K.; Agarwal, Amit

2015-01-01

Background: In the earlier studies, methanolic extract of Mangifera indica L leaf was exhibited hypocholesterol activity. However, the bioactive compounds responsible for the same are not reported so far. Objective: To isolate the bioactive compounds with hypocholesterol activity from the leaf extract using cholesterol esterase inhibition assay which can be used for the standardization of extract. Materials and Methods: The leaf methanolic extract of M. indica (Sindoora variety) was partitioned with ethyl acetate and chromatographed on silica gel to yield twelve fractions and the activity was monitored by using cholesterol esterase inhibition assay. Active fractions were re-chromatographed to yield individual compounds. Results and Discussion: A major compound mangiferin present in the extract was screened along with other varieties of mango leaves for cholesterol esterase inhibition assay. However, the result indicates that compounds other than mangiferin may be active in the extract. Invitro pancreatic cholesterol esterase inhibition assay was used for bioactivity guided fractionation (BAGF) to yield bioactive compound for standardization of extract. Bioactivity guided fractionation afford the active fraction containing 3b-taraxerol with an IC50 value of 0.86μg/ml. Conclusion: This study demonstrates that M. indica methanol extract of leaf have significant hypocholesterol activity which is standardized with 3b-taraxerol, a standardized extract for hypocholesterol activity resulted in development of dietary supplement from leaves of Mangifera indica. PMID:26692750

Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea Brine Pool

KAUST Repository

Mohamed, Yasmine M.

2013-11-28

The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 65 C), halotolerant (maintains its activity in up to 4.5â€...M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.

Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea Brine Pool

KAUST Repository

Mohamed, Yasmine M.; Ghazy, Mohamed A.; Sayed, Ahmed; Ouf, Amged; El-Dorry, Hamza; Siam, Rania

2013-01-01

The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 65 C), halotolerant (maintains its activity in up to 4.5â€...M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

Science.gov (United States)

Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-08-01

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

B-type esterases in the snail Xeropicta derbentina: An enzymological analysis to evaluate their use as biomarkers of pesticide exposure

Energy Technology Data Exchange (ETDEWEB)

Laguerre, Christel [Universite d' Avignon et des Pays de Vaucluse, UMR 406 UAPV/INRA, F-84914 Avignon (France); INRA, Laboratoire de Toxicologie Environnementale, UMR 406 UAPV/INRA, F-84914 Avignon (France); Sanchez-Hernandez, Juan C. [Laboratory of Ecotoxicology, Faculty of Environmental Science, University of Castilla-La Mancha, Avda. Carlos III s/n, 45071 Toledo (Spain); Koehler, Heinz R. [Animal Physiological Ecology, University of Tuebingen, Konrad-Adenauer-Strasse 20, D-72072 Tuebingen (Germany); Triebskorn, Rita [Animal Physiological Ecology, University of Tuebingen, Konrad-Adenauer-Strasse 20, D-72072 Tuebingen (Germany); Steinbeis-Transfer Center for Ecotoxicology and Ecophysiology, Blumenstrasse 13, D-72108 Rottenburg (Germany); Capowiez, Yvan [INRA, Unite PSH, F- 84914 Avignon (France); Rault, Magali [Universite d' Avignon et des Pays de Vaucluse, UMR 406 UAPV/INRA, F-84914 Avignon (France); INRA, Laboratoire de Toxicologie Environnementale, UMR 406 UAPV/INRA, F-84914 Avignon (France); Mazzia, Christophe [Universite d' Avignon et des Pays de Vaucluse, UMR 406 UAPV/INRA, F-84914 Avignon (France); INRA, Laboratoire de Toxicologie Environnementale, UMR 406 UAPV/INRA, F-84914 Avignon (France)], E-mail: mazzia@avignon.inra.fr

2009-01-15

The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (K{sub m} = 77.2 mM; V{sub max} = 38.2 mU/mg protein) and 1-naphthyl acetate (K{sub m} = 222 mM, V{sub max} = 1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50 = 1.35 x 10{sup -5}-3.80 x 10{sup -8} M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50 = 1.20 x 10{sup -5}-2.98 x 10{sup -8} M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions. - Characterization of the B-type esterases in the terrestrial snail Xeropicta derbentina in order to evaluate pesticide exposure.

Research of small quaternary AChE inhibitors as pretreatment of OP poisoning

International Nuclear Information System (INIS)

Musilek, K.; Komloova, M.; Holas, O.; Opletalova, V.; Pohanka, M.; Kuca, K.

2009-01-01

Small quaternary AChE inhibitors are used (e.g. pyridostigmine) or scoped (e.g. SAD-128) for pretreatment against organophosphate intoxication [1]. The pretreatment is based on competitive inhibition of AChE prior to organophosphate (OP) poisoning. Consequently, the OP can not influence the inhibited AChE and is degraded by other esterases. Although various competitive inhibitors are used globally, pyridostigmine still remains the most broaden. Its side effects including gastrointestinal effects (nausea, intestinal obstruction), increased bronchial secretion, cardiac arrhythmia or cholinergic crisis are well described. Moreover, some bisquaternary competitive inhibitors (e.g. SAD-128) were used to decrease lethal effects of OP poisoning in vivo. The further studies dealing with SAD-128 showed its increased ability to interact with brain muscarinic acetylcholine receptors as allosteric inhibitors [2]. The small molecules derived from quaternized pyridine, quinoline and isoquinoline were designed as AChE inhibitors. Their ability to inhibit AChE or BChE was determined in vitro using IC50. The IC50 data were compared within each group of compounds with emphasis on selectivity AChE versus BChE. The overall study will be presented. The work was supported by Ministry of Defence of Czech Republic No. OVUOFVZ200805.(author)

Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils.

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Yamamoto-Tamura, Kimiko; Hiradate, Syuntaro; Watanabe, Takashi; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Yarimizu, Tohru; Kitamoto, Hiroko

2015-01-01

The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these soils.

Esterase-D and chromosome patterns in Central Amazon piranha (Serrasalmus rhombeus Linnaeus, 1766 from Lake Catalão

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Aylton Saturnino Teixeira

2006-01-01

Full Text Available This study presents additional genetic data on piranha (Serrasalmus rhombeus Linnaeus, 1766 complex previously diagnosed due to the presence of distinct cytotypes 2n = 58 and 2n = 60. Three esterase-D enzyme loci (Est-D1, Est-D2 and Est-D3 were examined and complemented with chromosomal data from 66 piranha specimens collected from Lake Catalão. For all specimens the Est-D1 and Est-D2 loci were monomorphic. In contrast, the Est-D3 locus was polymorphic with genotypes and alleles being differentially distributed in the previously described cytotypes and served as the basis for detecting a new cytotype (2n = 60 B. In cytotype 2n = 58 the Est-D3 locus was also polymorphic and presented Mendelian allelic segregation with four genotypes (Est-D3(11, Est-D3(12, Est-D3(22 and Est-D3(33 out of six theoretically possible genotypes, presumably encoded by alleles Est-D3¹ (frequency = 0.237, EsT-D3² (0.710 and Est-D3³ (0.053. A Chi-squared (chi2 test for Hardy-Weinberg equilibrium was applied to the Est-D3 locus and revealed a genetic unbalance in cytotype 2n = 58, indicating the probable existence in the surveyed area of different stocks for that karyotypic structure. A silent null allele (Est-D3(0 with a high frequency (0.959 occurred exclusively in the 2n = 60 cytotype. On the other hand, the new cytotype 2n = 60 B described here for the first time was monomorphic for the presumably fixed Est-D3³ allele. The data as a whole should contribute to the better understanding the rhombeus complex taxonomic status definition in the Central Amazon.

Effects of piperonyl butoxide on the toxicity of the organophosphate temephos and the role of esterases in the insecticide resistance of Aedes aegypti

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Boscolli Barbosa Pereira

2014-10-01

Full Text Available Introduction The effects of piperonyl butoxide (PBO on the toxicity of the organophosphate temephos (TE and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated. Methods A. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined. Results At concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only. Conclusions The results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae.

Hepatitis C virus inhibitor synergism suggests multistep interactions between heat-shock protein 90 and hepatitis C virus replication

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Kubota, Naoko; Nomoto, Masataka; Hwang, Gi-Wook; Watanabe, Toshihiko; Kohara, Michinori; Wakita, Takaji; Naganuma, Akira; Kuge, Shusuke

2016-01-01

AIM: To address the effect of heat-shock protein 90 (HSP90) inhibitors on the release of the hepatitis C virus (HCV), a cell culture-derived HCV (JFH1/HCVcc) from Huh-7 cells was examined. METHODS: We quantified both the intracellular and extracellular (culture medium) levels of the components (RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A (CsA) or interferon. Finally, we statistically examined the combined effect of radicicol and CsA using the combination index (CI) and graphical representation proposed by Chou and Talalay. RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor (CsA or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy (CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release. PMID:26925202

EVALUATION OF ESTERASE POLYMORPHISMS IN MATURE SEEDS OF RADISH (RAPHANUS SATIVUS L. ACCESSIONS OF VIR COLLECTION

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A. S. Rudakova

2017-01-01

Full Text Available A biochemical evaluation of 25 radish accessions (Raphanus sativus L. on esterase isozymes of mature seeds has been carried out. The results of the experiments showed a wide range of diversity among the genotypes based on electrophoretic zones of esterase isoenzymes. The revealed isoenzyme complex of esterases was represented by eight isoforms with molecular weights from 37.7 kD to 57.6 kD. All accessions were divided into 13 electrophoretic zymotypes, differing from each other by the presence or absence of definite zones. The most often observed electrophoretic zymo-type is Gr. 1, which includes 24% of the total number of accessions evaluated. There are 8 zymotypes (Gr. 6 Gr. 13 with a frequency of occurrence 4%. Three groups (Gr. 2 – Gr. 4 had the same frequency of occurrence – 12%. Zimotype of Gr. 5 containes the maximum number of zones – 8. 2 zimotypes – Gr. 3 and Gr. 12 had the smallest number of 4 zones. Two zones of esterases – zones 7 and 8 (Мr 39.7кD and Мr 37.7 kD, respectively were monomorphic. The remaining six zones were polymorphic, i.e. could be absent in some zimotypes. The frequency of occurrence of each zone in different zymotypes has varied from 6.58% to 17.11%. As results of this research the accessions that were selected can become the most promising parent forms for future genetic and selection studies of this culture.

Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

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Orit Wolstein

2014-01-01

Full Text Available Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC. CCR5-targeted shRNA (sh5 and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system

Energy Technology Data Exchange (ETDEWEB)

Mangas, Iris; Vilanova, Eugenio; Estevez, Jorge, E-mail: jorge.estevez@umh.es

2011-11-15

Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and 'ongoing inhibition') of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and the model applied and may be considered an external validation. The most sensitive component (E{alpha}) for paraoxon (11-23% of activity, I{sub 50} (30 min) = 9-11 nM) is also the most sensitive for mipafox (I{sub 50} (30 min) = 4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (E{beta}, 71-84% of activity; I{sub 50} (30 min) = 1216 nM) is practically resistant to mipafox. The third component (E{gamma}, 5-8% of activity) is paraoxon resistant and has I{sub 50} (30 min) of 3.4 {mu}M with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies. -- Research Highlights: Black-Right-Pointing-Pointer Paraoxon and mipafox interactions have been evaluated with chicken soluble brain esterases. Black-Right-Pointing-Pointer The paraoxon inhibition was analyzed considering the simultaneous spontaneous reactivation. Black

Spatial distribution and esterase activity in populations of Aedes (Stegomyia aegypti (Linnaeus (Diptera: Culicidae resistant to temephos

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Wanessa Porto Tito Gambarra

2013-04-01

Full Text Available INTRODUCTION: The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypti collected in municipalities of the State of Paraíba. METHODS: Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector. RESULTS: All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR greater than 20. The greatest lethal dose 50% of the sample (CL50 was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and β-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity. CONCLUSIONS: Aedes aegypti is widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos.

Plasma B-esterase activities in European raptors.

Science.gov (United States)

Roy, Claudie; Grolleau, Gérard; Chamoulaud, Serge; Rivière, Jean-Louis

2005-01-01

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and

Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

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Patick, A K; Boritzki, T J; Bloom, L A

1997-10-01

Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity.

Synthesis of 1-benzyl-4-[(5,6-dimethoxy[2-14C]-1-indanon)-2-YL]-methylpiperidine hydrochloride (E2020-14C)

International Nuclear Information System (INIS)

Iimura, Youichi; Mishima, Mannen; Sugimoto, Hachiro

1989-01-01

1-Benzyl-4-[(5,6-dimethoxy[2- 14 C]-1-indanon)-2-yl]-methylpiperidine hydrochloride (E2020- 14 C), and acetylcholinesterase inhibitor for studying the pharmacokinetic profiles of E2020, was synthesized from 5,6-dimethoxy[2- 14 C]-1-indanone as the labelled starting material. (author)

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

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Katsume, Asao; Tokunaga, Yuko; Hirata, Yuichi; Munakata, Tsubasa; Saito, Makoto; Hayashi, Hitohisa; Okamoto, Koichi; Ohmori, Yusuke; Kusanagi, Isamu; Fujiwara, Shinya; Tsukuda, Takuo; Aoki, Yuko; Klumpp, Klaus; Tsukiyama-Kohara, Kyoko; El-Gohary, Ahmed; Sudoh, Masayuki; Kohara, Michinori

2013-10-01

Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Esterase- and pH-responsive poly(β-amino ester)-capped mesoporous silica nanoparticles for drug delivery

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Fernando, Isurika R.; Ferris, Daniel P.; Frasconi, Marco; Malin, Dmitry; Strekalova, Elena; Yilmaz, M. Deniz; Ambrogio, Michael W.; Algaradah, Mohammed M.; Hong, Michael P.; Chen, Xinqi; Nassar, Majed S.; Botros, Youssry Y.; Cryns, Vincent L.; Stoddart, J. Fraser

2015-04-01

Gating of mesoporous silica nanoparticles (MSNs) with the stimuli-responsive poly(β-amino ester) has been achieved. This hybrid nanocarrier releases doxorubicin (DOX) under acidic conditions or in the presence of porcine liver esterase. The DOX loaded poly(β-amino ester)-capped MSNs reduce cell viability when tested on MDA-MB-231 human breast cancer cells.Gating of mesoporous silica nanoparticles (MSNs) with the stimuli-responsive poly(β-amino ester) has been achieved. This hybrid nanocarrier releases doxorubicin (DOX) under acidic conditions or in the presence of porcine liver esterase. The DOX loaded poly(β-amino ester)-capped MSNs reduce cell viability when tested on MDA-MB-231 human breast cancer cells. Electronic supplementary information (ESI) available: Experimental details relating to (i) the synthesis and characterisation of the surface-functionalised MSN and POL (ii) cargo-loading and release studies in solution, (iii) cellular internalisation of nanomaterials, and (iv) cell viability tests. See DOI: 10.1039/c4nr07443b

Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma

NARCIS (Netherlands)

Elisen, M. G.; von dem Borne, P. A.; Bouma, B. N.; Meijers, J. C.

1998-01-01

Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited

Peptide Inhibitor of Complement C1 (PIC1 Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

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Julia A Sharp

Full Text Available The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1. In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

Characterization and distribution of esterase activity in activated sludge

NARCIS (Netherlands)

Boczar, BA; Forney, LJ; Begley, WM; Larson, RJ; Federle, TW

2001-01-01

The location and activity of esterase enzymes in activated Sludge from three Municipal wastewater treatment plants were characterized using model Substrate, and denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE) Of particulate, freeze thaw (primarily periplasmic enzymes and those

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

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Charlotte Kürten

2016-06-01

Full Text Available The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

Science.gov (United States)

Rao, Lang; Zhao, Xiubo; Pan, Fang; Li, Yin; Xue, Yanfen; Ma, Yanhe; Lu, Jian R.

2009-01-01

Background Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that

Functional characterization of salt-tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)-3-hydroxybutyrate.

Science.gov (United States)

Wang, Yilong; Xu, Yongkai; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

2018-06-01

The two enantiomers of ethyl 3-hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)-3-hydroxybutyrate. Herein, we also functionally characterized one novel salt-tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)-3-hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio-selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates. © 2018 Wiley Periodicals, Inc.

Leucocyte esterase in the rapid diagnosis of paediatric septic arthritis.

LENUS (Irish Health Repository)

Kelly, E G

2013-02-01

Septic arthritis may affect any age group but is more common in the paediatric population. Infection is generally bacterial in nature. Prompt diagnosis is crucial, as delayed treatment is associated with lifelong joint dysfunction. A clinical history and application of Kocher\\'s criteria may indicate that there is a septic arthritis. However, definitive diagnosis is made on culture of septic synovial fluid. The culture process can take over 24h for the initial culture to yield bacterial colonies. Leucocyte esterase is released by leucocytes at the site of an infection. We hypothesise that leucocyte esterase can be utilized in the rapid diagnosis of septic arthritis and shorten the time to decisive treatment whilst simultaneously decreasing unnecessary treatment of non-septic joints.

Evidence-based recommendations for the therapeutic management of angioedema owing to hereditary C1 inhibitor deficiency: consensus report of an International Working Group

NARCIS (Netherlands)

Cicardi, M.; Bork, K.; Caballero, T.; Craig, T.; Li, H. H.; Longhurst, H.; Reshef, A.; Zuraw, B.; Werner, Aberer; Aygören-Pürsün, Emel; Banerji, Aleena; Bjorkander, Janne; Boccon-Gibod, Isabelle; Konrad, Bork; Bouillet, Laurence; Bova, Maria; Bowen, Tom; Branco Ferreira, Manuel; Bygum, Anette; Caballero, Teresa; Cancian, Mauro; Castel-Branco, Maria Graça; Cicardi, Marco; Craig, Timothy; de Carolis, Caterina; Mihály, Enikö; Josè, Fabiani; Farkas, Henriette; Gompels, Mark; Gower, Richard; Groffik, Adriane; Grumach, Anete; Guillarte, Mar; Hernandez Landeros, Maria Esthela; Kaplan, Allen; Leibovich, Iris; Li, Henry; Lock, Bob; Longhurst, Hilary; Lumry, William; Malbran, Alejandro; Martinez-Saguer, Immaculada; Campos, Matta; Maurer, Marcus; Moldovan, Dumitru; Montinaro, Vincenzo; Nieto, Sandra; Nordenfelt, Patrik; Obtulovicz, Krystana; Zeerleder, Sacha

2012-01-01

Angioedema owing to hereditary deficiency of C1 inhibitor (HAE) is a rare, life-threatening, disabling disease. In the last 2 years, the results of well-designed and controlled trials with existing and new therapies for this condition have been published, and new treatments reached the market.

Enzymatic degradation of lignin‐carbohydrate complexes (LCCs): Model studies using a fungal glucuronoyl esterase from Cerrena unicolor

DEFF Research Database (Denmark)

d'Errico, Clotilde; Jørgensen, Jonas O.; Krogh, Kristian B. R. M.

2015-01-01

Lignin‐carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has...

A feruloyl esterase derived from a leachate metagenome library

CSIR Research Space (South Africa)

Rashamuse, K

2012-01-01

Full Text Available A feruloyl esterase encoding gene (designated fae6), derived from a leachate metagenomic library, was cloned and the nucleotide sequence of the insert DNA determined. Translational analysis revealed that fae6 consists of a 515 amino acid polypeptide...

Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

Science.gov (United States)

Pontikis, R; Dollé, V; Guillaumel, J; Dechaux, E; Note, R; Nguyen, C H; Legraverend, M; Bisagni, E; Aubertin, A M; Grierson, D S; Monneret, C

2000-05-18

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are favourable to glucagon-like peptide-1 (GLP-1) agonists

DEFF Research Database (Denmark)

Madsbad, Sten

2012-01-01

Incretin-based therapies, which include the GLP-1 receptor agonists and DPP-4 inhibitors, use the antidiabetic properties of potentiating the GLP-1 receptor signalling via the regulation of insulin and glucagon secretion, inhibition of gastric emptying and suppression of appetite. Most physicians...... will start antidiabetic treatment with metformin, but adding a GLP-1 receptor agonist as the second drug seems to be optimal since more patients will reach an HbA1c below 7% than with a DPP-4 inhibitor or another oral antidiabetic agents and with minimal risk of hypoglycaemia. The GLP-1 receptor agonists...

Eco-friendly surface modification on polyester fabrics by esterase treatment

Energy Technology Data Exchange (ETDEWEB)

Wu, Jindan; Cai, Guoqiang; Liu, Jinqiang; Ge, Huayun; Wang, Jiping, E-mail: jipingwanghz@gmail.com

2014-03-01

Graphical abstract: - Highlights: • We used a simple and easy way to measure the enzyme activity. • We studied the mechanism by characterizing the chemical changes in the surface of fabric. • We studied the advantages in surface wettability, fiber integrity and mechanical performance of cutinase treated fabrics. • Cutinase pretreated fibers exhibited much improved fabric wicking and better fiber integrity comparing to alkali treated ones. • Cutinase pretreatment technology promotes energy conservation and emission reduction. - Abstract: Currently, traditional alkali deweighting technology is widely used to improve the hydrophilicity of polyester fabrics. However, the wastewater and heavy chemicals in the effluent cause enormous damage to the environment. Esterase treatment, which is feasible in mild conditions with high selectivity, can provide a clean and efficient way for polyester modification. Under the optimum conditions, the polyester fabric hydrolysis process of esterase had a linear kinetics. X-ray photoelectron spectrometry (XPS) results showed that hydroxyl and carboxyl groups were produced only on the surface of modified fiber without changing the chemical composition of the bulk. These fibers exhibited much improved fabric wicking, as well as greatly improved oily stain removal performance. Compared to the harsh alkali hydrolysis, the enzyme treatment led to smaller weight loss and better fiber integrity. The esterase treatment technology is promising to produce higher-quality polyester textiles with an environmental friendly approach.

Recent advances in the discovery of small molecule c-Met Kinase inhibitors.

Science.gov (United States)

Parikh, Palak K; Ghate, Manjunath D

2018-01-01

c-Met is a prototype member of a subfamily of heterodimeric receptor tyrosine kinases (RTKs) and is the receptor for hepatocyte growth factor (HGF). Binding of HGF to its receptor c-Met, initiates a wide range of cellular signalling, including those involved in proliferation, motility, migration and invasion. Importantly, dysregulated HGF/c-Met signalling is a driving factor for numerous malignancies and promotes tumour growth, invasion, dissemination and/or angiogenesis. Dysregulated HGF/c-Met signalling has also been associated with poor clinical outcomes and resistance acquisition to some approved targeted therapies. Thus, c-Met kinase has emerged as a promising target for cancer drug development. Different therapeutic approaches targeting the HGF/c-Met signalling pathway are under development for targeted cancer therapy, among which small molecule inhibitors of c-Met kinase constitute the largest effort within the pharmaceutical industry. The review is an effort to summarize recent advancements in medicinal chemistry development of small molecule c-Met kinase inhibitors as potential anti-cancer agents which would certainly help future researchers to bring further developments in the discovery of small molecule c-Met kinase inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Receptor tyrosine kinase (c-Kit inhibitors: a potential therapeutic target in cancer cells

Directory of Open Access Journals (Sweden)

Abbaspour Babaei M

2016-08-01

Full Text Available Maryam Abbaspour Babaei,1 Behnam Kamalidehghan,2,3 Mohammad Saleem,4–6 Hasniza Zaman Huri,1,7 Fatemeh Ahmadipour1 1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB, Shahrak-e Pajoohesh, 3Medical Genetics Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Urology, 5Department of Laboratory Medicine and Pathology, Masonic Cancer Center, University of Minnesota, 6Section of Molecular Therapeutics & Cancer Health Disparity, The Hormel Institute, Austin, MN, USA; 7Clinical Investigation Centre, University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia Abstract: c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c

Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection.

Science.gov (United States)

Côté, Marceline; Misasi, John; Ren, Tao; Bruchez, Anna; Lee, Kyungae; Filone, Claire Marie; Hensley, Lisa; Li, Qi; Ory, Daniel; Chandran, Kartik; Cunningham, James

2011-08-24

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

Pig Liver Esterase (PLE) as Biocatalyst in Organic Synthesis: From Nature to Cloning and to Practical Applications

NARCIS (Netherlands)

Dominguez de Maria, Pablo; Garcia-Burgos, Carlos A.; Bargeman, Gerrald; van Gemert, Robert W.

2007-01-01

Pig liver esterase (PLE, EC 3.1.1.1) has been employed extensively for research purposes during the last three decades, especially in kinetic resolutions, in desymmetrizations of prochiral substrates, and in the synthesis of nucleosides. Its practical use, however, has been traditionally hampered

Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes

International Nuclear Information System (INIS)

Ketas, Thomas J.; Schader, Susan M.; Zurita, Juan; Teo, Esther; Polonis, Victoria; Lu Min; Klasse, Per Johan; Moore, John P.

2007-01-01

Inhibitors of viral entry are under consideration as topical microbicides to prevent HIV-1 sexual transmission. Small molecules targeting HIV-1 gp120 (BMS-378806) or CCR5 (CMPD167), and a peptide fusion inhibitor (C52L), each blocks vaginal infection of macaques by a SHIV. A microbicide, however, must be active against multiple HIV-1 variants. We therefore tested BMS-C (a BMS-378806 derivative), CMPD167, C52L and the CXCR4 ligand AMD3465, alone and in combination, against 25 primary R5, 12 X4 and 7 R5X4 isolates from subtypes A-G. At high concentrations (0.1-1 μM), the replication of most R5 isolates in human donor lymphocytes was inhibited by > 90%. At lower concentrations, double and triple combinations were more effective than individual inhibitors. Similar results were obtained with X4 viruses when AMD3465 was substituted for CMPD167. The R5X4 viruses were inhibited by combining AMD3465 with CMPD167, or by the coreceptor-independent compounds. Thus, combining entry inhibitors may improve microbicide effectiveness

Synthesis of 14C analogue of 1,2-diaryl-[2-14C]-pyrroles

International Nuclear Information System (INIS)

Saemian, N.; Shirvani, G.; Matloubi, H.

2007-01-01

Three 1,2-diaryl pyrroles selective COX-2 inhibitors, 2-(4-fluorophenyl)-5-methyl-1-(4-methylsulfonyl-phenyl)-1H pyrrole, 2-(4-fluorophenyl)-1- [4-(methylsulfonyl) phenyl]-1H-pyrrole and 4-[2-(4-fluorophenyl)-1H-pyrrol-1-yl]benzenesulfon-amide, all three labeled with 14 C in the 2-position were prepared from para-fluoro-benzaldehyde-[carbonyl- 14 C]. (author)

Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase

Directory of Open Access Journals (Sweden)

Thomas Lanyon-Hogg

2016-06-01

Full Text Available In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI” class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a, RU-SKI 43 (9b, RU-SKI 101 (9c, and RU-SKI 201 (9d were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015 [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.

Activity of pectin methyl esterase during blanching of peaches

NARCIS (Netherlands)

Tijskens, L.M.M.; Rodis, P.S.; Hertog, M.L.A.T.M.; Proxenia, N.; Dijk, van C.

1999-01-01

The activity of pectin methyl esterase (PE) in peaches during blanching treatments was modelled and analyzed. It was postulated that the enzyme exists in two configurations, one bound and one soluble. The bound configuration can be converted into the soluble configuration. These two configurations

Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties.

Science.gov (United States)

Dyck, Brian; Branstetter, Bryan; Gharbaoui, Tawfik; Hudson, Andrew R; Breitenbucher, J Guy; Gomez, Laurent; Botrous, Iriny; Marrone, Tami; Barido, Richard; Allerston, Charles K; Cedervall, E Peder; Xu, Rui; Sridhar, Vandana; Barker, Ryan; Aertgeerts, Kathleen; Schmelzer, Kara; Neul, David; Lee, Dong; Massari, Mark Eben; Andersen, Carsten B; Sebring, Kristen; Zhou, Xianbo; Petroski, Robert; Limberis, James; Augustin, Martin; Chun, Lawrence E; Edwards, Thomas E; Peters, Marco; Tabatabaei, Ali

2017-04-27

A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.

The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch

DEFF Research Database (Denmark)

Mosbech, Caroline; Holck, Jesper; Meyer, Anne S.

2018-01-01

Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin-carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Here, we show...... for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo......-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. The data verify the enzyme's unique ability to catalyze removal of all glucuronoxylan associated...

In vivo evaluation of carbon-11-labelled non-sarcosine-based glycine transporter 1 inhibitors in mice and conscious monkeys

International Nuclear Information System (INIS)

Toyohara, Jun; Ishiwata, Kiichi; Sakata, Muneyuki; Wu, Jin; Nishiyama, Shingo; Tsukada, Hideo; Hashimoto, Kenji

2011-01-01

Introduction: Glycine transporter 1 (GlyT-1) is an attractive target in positron emission tomography (PET) studies. Here, we report the in vivo evaluation of three carbon-11-labelled non-sarcosine-type GlyT-1 inhibitors - [ 11 C]SA1, [ 11 C]SA2 and [ 11 C]SA3 - as novel PET tracers for GlyT-1. Methods: The regional brain distributions of the three compounds in mice were studied at baseline and under receptor-blockade conditions with co-injection of carrier loading or pretreatment with an excess of selective GlyT-1 inhibitors (ALX-5407 and SSR504734). Metabolic stability was investigated by radio high-performance liquid chromatography. Dynamic PET scans in conscious monkeys were performed with/without selective GlyT-1 inhibitors. Results: The IC 50 values of SA1, SA2 and SA3 were 9.0, 6400 and 39.7 nM, respectively. The regional brain uptakes of [ 11 C]SA1 and [ 11 C]SA3 in mice were heterogeneous and consistent with the known distribution of GlyT-1. [ 11 C]SA2 showed low and homogeneous uptake in the brain. Most radioactivity in the brain was detected in unchanged form, although peripherally these compounds were degraded. Carrier loading decreased the uptake of [ 11 C]SA1 in GlyT-1-rich regions. However, similar reductions were not observed with [ 11 C]SA3. Pretreatment with ALX-5407 decreased the uptake of [ 11 C]SA1 in GlyT-1-rich regions. In the monkey at baseline, regional brain uptake of [ 11 C]SA1 was heterogeneous and consistent with the known GlyT-1 distribution. Pretreatment with selective GlyT-1 inhibitors significantly decreased the distribution volume ratio of [ 11 C] SA1 in GlyT-1-rich regions. Conclusions: [ 11 C]SA1 has the most suitable profile among the three carbon-11-labelled GlyT-1 inhibitors. Lead optimization of [ 11 C]SA1 structure will be required to achieve in vivo selective GlyT-1 imaging.

Virtual screening for potential inhibitors of bacterial MurC and MurD ligases.

Science.gov (United States)

Tomašić, Tihomir; Kovač, Andreja; Klebe, Gerhard; Blanot, Didier; Gobec, Stanislav; Kikelj, Danijel; Mašič, Lucija Peterlin

2012-03-01

Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.

DPP-4 inhibitor treatment: β-cell response but not HbA1c reduction is dependent on the duration of diabetes

Directory of Open Access Journals (Sweden)

Kozlovski P

2017-03-01

Full Text Available Plamen Kozlovski,1 Vaishali Bhosekar,2 James E Foley3 1Novartis Pharma AG, Basel, Switzerland; 2Novartis Healthcare Private Limited, Hyderabad, India; 3Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA Introduction: Dipeptidyl peptidase-4 (DPP-4 inhibitors reduce hyperglycemia in patients with type 2 diabetes mellitus (T2DM by enhancing insulin and suppressing glucagon secretion. Since T2DM is associated with progressive loss of β-cell function, we hypothesized that the DPP-4 inhibitor action to improve β-cell function would be attenuated with longer duration of T2DM.Methods: Data from six randomized, placebo-controlled trials of 24 weeks duration, where β-cell response to vildagliptin 50 mg twice daily was assessed, were pooled. In each study, the insulin secretory rate relative to glucose (ISR/G 0–2h during glucose load (standard meal or oral glucose tolerance test was assessed at baseline and end of study. The mean placebo-subtracted difference (PSD in the change in ISR/G 0–2h from baseline for each study was evaluated as a function of age, duration of T2DM, baseline ISR/G 0–2h, glycated hemoglobin (HbA1c, fasting plasma glucose, body mass index, and mean PSD in the change in HbA1c from baseline, using univariate model.Results: There was a strong negative association between the PSD in the change from baseline in ISR/G 0–2h and duration of T2DM (r= −0.89, p<0.02. However, there was no association between the PSD in the change from baseline in ISR/G 0–2h and the PSD in the change from baseline in HbA1c (r=0.33, p=0.52. None of the other characteristics were significantly associated with mean PSD change in ISR/G 0–2h.Conclusion: These findings indicate that the response of the β-cell, but not the HbA1c reduction, with vildagliptin is dependent on duration of T2DM. Further, it can be speculated that glucagon suppression may become the predominant mechanism via which glycemic control is improved when treatment with a

Esterase-sensitive sulfur dioxide prodrugs inspired by modified Julia olefination.

Science.gov (United States)

Wang, Wenyi; Wang, Binghe

2017-09-12

Sulfur dioxide (SO 2 ) is an endogenously produced gaseous molecule, and is emerging as a potential gasotransmitter. Herein, we describe the first series of esterase-sensitive prodrugs inspired by modified Julia olefination as SO 2 donors.

Safety and Usage of C1-Inhibitor in Hereditary Angioedema

DEFF Research Database (Denmark)

Riedl, Marc A; Bygum, Anette; Lumry, William

2016-01-01

, international patient registry documented widespread implementation of pnfC1-INH self-administration outside of a health care setting consistent with current HAE guidelines. These real-world data revealed pnfC1-INH usage for a variety of reasons in patients with HAE and showed a high level of safety regardless...... of this study was to describe safety and usage patterns of pnfC1-INH. METHODS: A multicenter, observational, registry was conducted between 2010 and 2014 at 30 United States and 7 European sites to obtain both prospective (occurring after enrollment) and retrospective (occurring before enrollment) safety...... and usage data on subjects receiving pnfC1-INH for any reason. RESULTS: Of 343 enrolled patients, 318 received 1 or more doses of pnfC1-INH for HAE attacks (11,848 infusions) or for prophylaxis (3142 infusions), comprising the safety population. Median dosages per infusion were 10.8 IU/kg (attack treatment...

Connexin31.1 deficiency in the mouse impairs object memory and modulates open-field exploration, acetylcholine esterase levels in the striatum, and cAMP response element-binding protein levels in the striatum and piriform cortex.

Science.gov (United States)

Dere, E; Zheng-Fischhöfer, Q; Viggiano, D; Gironi Carnevale, U A; Ruocco, L A; Zlomuzica, A; Schnichels, M; Willecke, K; Huston, J P; Sadile, A G

2008-05-02

Neuronal gap junctions in the brain, providing intercellular electrotonic signal transfer, have been implicated in physiological and behavioral correlates of learning and memory. In connexin31.1 (Cx31.1) knockout (KO) mice the coding region of the Cx31.1 gene was replaced by a LacZ reporter gene. We investigated the impact of Cx31.1 deficiency on open-field exploration, the behavioral response to an odor, non-selective attention, learning and memory performance, and the levels of memory-related proteins in the hippocampus, striatum and the piriform cortex. In terms of behavior, the deletion of the Cx31.1 coding DNA in the mouse led to increased exploratory behaviors in a novel environment, and impaired one-trial object recognition at all delays tested. Despite strong Cx31.1 expression in the peripheral and central olfactory system, Cx31.1 KO mice exhibited normal behavioral responses to an odor. We found increased levels of acetylcholine esterase (AChE) and cAMP response element-binding protein (CREB) in the striatum of Cx31.1 KO mice. In the piriform cortex the Cx31.1 KO mice had an increased heterogeneity of CREB expression among neurons. In conclusion, gap-junctions featuring the Cx31.1 protein might be involved in open-field exploration as well as object memory and modulate levels of AChE and CREB in the striatum and piriform cortex.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

Science.gov (United States)

Nishiura, T; Abe, K

1999-01-01

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

Carbon-11 labelling of an inhibitor of acetylcholinesterase: [11C]physostigmine

International Nuclear Information System (INIS)

Bonnot-Lours, S.; Crouzel, C.; Prenant, C.; Hinnen, F.

1993-01-01

Physostigmine, an alkaloid from calabar bean is a strong inhibitor of acetylcholinesterase and has been used clinically in the treatment of glaucoma, atropine intoxication, myasthenia gravis and more recently, in experimental trials in Alzheimer's disease. In order to study the AChE activity in the brain by positron emission tomography, we have undertaken the labelling of physostigmine with carbon-11. The synthesis involves the reaction of [ 11 C]methylisocyanate with eseroline. [ 11 C]Methylisocyanate was obtained by heating [ 11 C]acetylchloride with tetrabutylammonium azide in toluene. The synthesis of [ 11 C]CH 3 COC1 involves the carbonation of methylmagnesium bromide in THF with cyclotron produced [ 11 C]carbon dioxide and the addition of phthaloyl dichloride. The [ 11 C]methylisocyanate is distilled into a solution of eseroline in ether with a small piece of sodium. After 10 minutes at 25 o C, the solution is purified by HPLC and the appropriate fraction collected. Starting with 55.5 GBq (1.5 Ci) of [ 11 C]carbon dioxide, 0.92-1.48 GBq (25-40 mCi) of [ 11 C]Physostigmine are obtained 57 minutes after EOB. (author)

Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1.

Science.gov (United States)

Guo, L-J; Luo, X-H; Xie, H; Zhou, H-D; Yuan, L-Q; Wang, M; Liao, E-Y

2006-05-01

We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

Science.gov (United States)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

2010-04-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

Energy Technology Data Exchange (ETDEWEB)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J.; Madrid, Susan M.; Brinch-Pedersen, Henrik; Holm, Preben B.; Scheller, Henrik V.

2009-12-08

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Structural insights into the binding mechanism of IDO1 with hydroxylamidine based inhibitor INCB14943

International Nuclear Information System (INIS)

Wu, You; Xu, Tingting; Liu, Jinsong; Ding, Ke; Xu, Jinxin

2017-01-01

IDO1 (indoleamine 2, 3-dioxygenase 1), a well characterized immunosuppressive enzyme, has attracted growing attention as a potential target for cancer immunotherapy. Hydroxylamidine compounds INCB024360 and INCB14943 (INCB024360 analogue) are highly effective IDO1 inhibitors. INCB024360 is undergoing clinical trials for treatment of various types of human cancer. Here, we determined the co-crystal structure of IDO1 and INCB14943, and elucidate the detailed binding mode. INCB14943 binds to heme iron in IDO1 protein through the oxime nitrogen. Further analysis also reveals that a halogen bonding interaction between the chlorine atom (3-Cl) of INCB14943 and the sulphur atom of C129 significantly improves the inhibition activity against IDO1. Comparing with the other reported inhibitors, the oxime nitrogen and halogen bond interaction are identified as the unique features of INCB14943 among the IDO1 inhibitors. Thus, our study provides novel insights into the interaction between a small molecule inhibitor INCB14943 and IDO1 protein. The structural information will facilitate future IDO1 inhibitor design. - Highlights: • This is the first co-crystal structure of IDO1 with hydroxylamidine compound. • INCB14943 binds to heme iron through oxime nitrogen instead of imidazole nitrogen. • Halogen bond interaction with C129 is another unique feature of INCB14943.

Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

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Guanhua Du

2011-12-01

Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

CD38 Structure-Based Inhibitor Design Using the N1-Cyclic Inosine 5'-Diphosphate Ribose Template.

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Christelle Moreau

Full Text Available Few inhibitors exist for CD38, a multifunctional enzyme catalyzing the formation and metabolism of the Ca(2+-mobilizing second messenger cyclic adenosine 5'-diphosphoribose (cADPR. Synthetic, non-hydrolyzable ligands can facilitate structure-based inhibitor design. Molecular docking was used to reproduce the crystallographic binding mode of cyclic inosine 5'-diphosphoribose (N1-cIDPR with CD38, revealing an exploitable pocket and predicting the potential to introduce an extra hydrogen bond interaction with Asp-155. The purine C-8 position of N1-cIDPR (IC50 276 µM was extended with an amino or diaminobutane group and the 8-modified compounds were evaluated against CD38-catalyzed cADPR hydrolysis. Crystallography of an 8-amino N1-cIDPR:CD38 complex confirmed the predicted interaction with Asp-155, together with a second H-bond from a realigned Glu-146, rationalizing the improved inhibition (IC50 56 µM. Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 µM with CD38 illustrated that Glu-146 hydrogen bonds with the ligand N6-amino group. Both 8-amino N1-cIDPR and cADPcR bind deep in the active site reaching the catalytic residue Glu-226, and mimicking the likely location of cADPR during catalysis. Substantial overlap of the N1-cIDPR "northern" ribose monophosphate and the cADPcR carbocyclic ribose monophosphate regions suggests that this area is crucial for inhibitor design, leading to a new compound series of N1-inosine 5'-monophosphates (N1-IMPs. These small fragments inhibit hydrolysis of cADPR more efficiently than the parent cyclic compounds, with the best in the series demonstrating potent inhibition (IC50 = 7.6 µM. The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design.

14C-labeling of a tetrahydroacridine, a novel CNS-selective cholinesterase inhibitor

International Nuclear Information System (INIS)

Nishioka, Kazuhiko; Kamada, Takeshi; Kanamaru, Hiroshi

1992-01-01

9-Amino-8-fluoro-2,4-methano-1,2,3,4-tetrahydroacridine citrate (SM-10888), a novel cholinesterase inhibitor, was labeled with carbon-14 at C9 of the tetrahydroacridine ring for use in metabolic studies. Carbonation of 2,6-difluorophenyllithium (3) with [ 14 C]carbon dioxide gave the acid (4). Chlorination of 4 followed by treatment of the resulting acid chloride with ammonia afforded the amide (5). Dehydration of 5 with thionyl chloride and subsequent displacement reaction with ammonia gave the aminobenzonitrile (7). Condensation of 7 with the ketone (8) in the presence of anhydrous zinc chloride yielded the aminoacridine (9), which was treated with citric acid to afford [9- 14 C]SM-10888 (1). The overall yield of 1 was 37% from 2, and the specific activity was 1.35 GBq/mmol. (author)

HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

Science.gov (United States)

Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D; Haughn, George W

2015-03-01

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy. © 2015 American Society of Plant Biologists. All Rights Reserved.

Esterases of laboratory-reared and field-collected cotton boll weevils, Anthonomus grandis Boh.: polymorphism of adult esterases and formal genetics of esterase II.

Science.gov (United States)

Biggers, C J; Bancroft, H R

1977-04-01

The esterases of the cotton boll weevil were separated by polyacrylamide gel electrophoresis into four major regions. These were named Est I-IV in order of migration from anode to origin. Polymorphism was observed in all regions. The Est II region was shown to consist of no more than two bands (fast and slow). The inheritance of the fast and slow bands of Est II was demonstrated to be controlled by codominant autosomal alleles. Analysis of the gene frequency of the Est II region showed that one field population was consistent with the Hardy-Weinberg law (P = 0.995), while a second field population was not at equilibrium (P less than 0.001).

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

Science.gov (United States)

Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

2016-04-01

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Heterologous expression of two Aspergillus niger feruloyl esterases in Trichoderma reesei for the production of ferulic acid from wheat bran.

Science.gov (United States)

Long, Liangkun; Zhao, Haoyuan; Ding, Dafan; Xu, Meijuan; Ding, Shaojun

2018-05-01

Feruloyl esterase (FAE)-encoding genes AnfaeA and AnfaeB were isolated from Aspergillus niger 0913. For overexpression of the two genes in Trichoderma reesei, constitutive and inductive expression plasmids were constructed based on parental plasmid pAg1-H3. The constructed plasmids contained AnfaeA or AnfaeB gene under the control of glyceraldehyde-3-phosphate dehydrogenase A gene (gpdA) promoter (from A. nidulans) or cellobiohydrolases I (cbh I) gene promoter (from T. reesei), and cbh I terminator from T. reesei. The target plasmids were transferred into T. reesei D-86271 (Rut-C30) by Agrobacterium tumefaciens-mediated transformation (ATMT), respectively. A high level of feruloyl esterase was produced by the recombinant fungal strains under solid-state fermentation, and the cbh I promoter was more efficient than the gpdA promoter in the expression of AnfaeA. The optimum temperatures and pH values were 50 °C and 5.0 for AnFAEA, and 35 °C and 6.0 for AnFAEB. The maximum production levels were 20.69 U/gsd for AnFAEA and 15.08 U/gsd for AnFAEB. The recombinant fungal enzyme systems could release 62.9% (for AnFAEA) and 52.2% (for AnFAEB) of total ferulic acids from de-starched wheat bran, which was higher than the 46.3% releasing efficiency of A. niger 0913. The supplement of xylanase from T. longibrachiatum in the enzymatic hydrolysis led to a small increment of the ferulic acids release.

Synthesis of 1-benzyl-4-((5,6-dimethoxy(2- sup 14 C)-1-indanon)-2-YL)-methylpiperidine hydrochloride (E2020- sup 14 C)

Energy Technology Data Exchange (ETDEWEB)

Iimura, Youichi; Mishima, Mannen; Sugimoto, Hachiro (Eisai Co., Ltd., Ibaraki (Japan). Tsukuba Research Labs.)

1989-07-01

1-Benzyl-4-((5,6-dimethoxy(2-{sup 14}C)-1-indanon)-2-yl)-methylpiperidine hydrochloride (E2020-{sup 14}C), and acetylcholinesterase inhibitor for studying the pharmacokinetic profiles of E2020, was synthesized from 5,6-dimethoxy(2-{sup 14}C)-1-indanone as the labelled starting material. (author).

C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

Science.gov (United States)

Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

2004-01-30

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing.

Science.gov (United States)

Reader, John C; Matthews, Thomas P; Klair, Suki; Cheung, Kwai-Ming J; Scanlon, Jane; Proisy, Nicolas; Addison, Glynn; Ellard, John; Piton, Nelly; Taylor, Suzanne; Cherry, Michael; Fisher, Martin; Boxall, Kathy; Burns, Samantha; Walton, Michael I; Westwood, Isaac M; Hayes, Angela; Eve, Paul; Valenti, Melanie; de Haven Brandon, Alexis; Box, Gary; van Montfort, Rob L M; Williams, David H; Aherne, G Wynne; Raynaud, Florence I; Eccles, Suzanne A; Garrett, Michelle D; Collins, Ian

2011-12-22

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

Hepatitis C virus protease inhibitor-resistance mutations: our experience and review.

Science.gov (United States)

Wu, Shuang; Kanda, Tatsuo; Nakamoto, Shingo; Imazeki, Fumio; Yokosuka, Osamu

2013-12-21

Direct-acting antiviral agents (DAAs) for hepatitis C virus (HCV) infection are one of the major advances in its medical treatment. The HCV protease inhibitors boceprevir and telaprevir were the first approved DAAs in the United States, Europe, and Japan. When combined with peginterferon plus ribavirin, these agents increase sustained virologic response rates to 70%-80% in treatment-naïve patients and previous-treatment relapsers with chronic HCV genotype 1 infection. Without peginterferon plus ribavirin, DAA mono-therapies increased DAA-resistance mutations. Several new DAAs for HCV are now in clinical development and are likely to be approved in the near future. However, it has been reported that the use of these drugs also led to the emergence of DAA-resistance mutations in certain cases. Furthermore, these mutations exhibit cross-resistance to multiple drugs. The prevalence of DAA-resistance mutations in HCV-infected patients who were not treated with DAAs is unknown, and it is as yet uncertain whether such variants are sensitive to DAAs. We performed a population sequence analysis to assess the frequency of such variants in the sera of HCV genotype 1-infected patients not treated with HCV protease inhibitors. Here, we reviewed the literature on resistance variants of HCV protease inhibitors in treatment naïve patients with chronic HCV genotype 1, as well as our experience.

HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

DEFF Research Database (Denmark)

Vanangamudi, Murugesan; Poongavanam, Vasanthanathan; Namasivayam, Vigneshwaran

2017-01-01

BACKGROUND: Design of inhibitors for HIV-1 reverse transcriptase inhibition (HIV-1 RT) is one of the successful chemotherapies for the treatment of HIV infection. Among the inhibitors available for HIV-1 RT, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have shown to be very promising......: The conformation dependent-alignment based (CoMFA and CoMSIA) methods have been proven very successful ligand based strategy in the drug design. Here, CoMFA and CoMSIA studies reported for structurally distinct NNRTIs including thiazolobenzimidazole, dipyridodiazepinone, 1,1,3-trioxo [1,2,4]-thiadiazine...

Taxane-Platin-Resistant Lung Cancers Co-develop Hypersensitivity to JumonjiC Demethylase Inhibitors

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Maithili P. Dalvi

2017-05-01

Full Text Available Although non-small cell lung cancer (NSCLC patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.

In vivo evaluation of carbon-11-labelled non-sarcosine-based glycine transporter 1 inhibitors in mice and conscious monkeys

Energy Technology Data Exchange (ETDEWEB)

Toyohara, Jun [Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan 260-8670 (Japan); Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan 173-0022 (Japan); Ishiwata, Kiichi; Sakata, Muneyuki [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan 173-0022 (Japan); Wu, Jin [Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan 260-8670 (Japan); Nishiyama, Shingo; Tsukada, Hideo [Central Research Laboratory, Hamamatsu Photonics K.K., Shizuoka, Japan 434-8601 (Japan); Hashimoto, Kenji, E-mail: hashimoto@faculty.chiba-u.j [Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan 260-8670 (Japan)

2011-05-15

Introduction: Glycine transporter 1 (GlyT-1) is an attractive target in positron emission tomography (PET) studies. Here, we report the in vivo evaluation of three carbon-11-labelled non-sarcosine-type GlyT-1 inhibitors - [{sup 11}C]SA1, [{sup 11}C]SA2 and [{sup 11}C]SA3 - as novel PET tracers for GlyT-1. Methods: The regional brain distributions of the three compounds in mice were studied at baseline and under receptor-blockade conditions with co-injection of carrier loading or pretreatment with an excess of selective GlyT-1 inhibitors (ALX-5407 and SSR504734). Metabolic stability was investigated by radio high-performance liquid chromatography. Dynamic PET scans in conscious monkeys were performed with/without selective GlyT-1 inhibitors. Results: The IC{sub 50} values of SA1, SA2 and SA3 were 9.0, 6400 and 39.7 nM, respectively. The regional brain uptakes of [{sup 11}C]SA1 and [{sup 11}C]SA3 in mice were heterogeneous and consistent with the known distribution of GlyT-1. [{sup 11}C]SA2 showed low and homogeneous uptake in the brain. Most radioactivity in the brain was detected in unchanged form, although peripherally these compounds were degraded. Carrier loading decreased the uptake of [{sup 11}C]SA1 in GlyT-1-rich regions. However, similar reductions were not observed with [{sup 11}C]SA3. Pretreatment with ALX-5407 decreased the uptake of [{sup 11}C]SA1 in GlyT-1-rich regions. In the monkey at baseline, regional brain uptake of [{sup 11}C]SA1 was heterogeneous and consistent with the known GlyT-1 distribution. Pretreatment with selective GlyT-1 inhibitors significantly decreased the distribution volume ratio of [{sup 11}C] SA1 in GlyT-1-rich regions. Conclusions: [{sup 11}C]SA1 has the most suitable profile among the three carbon-11-labelled GlyT-1 inhibitors. Lead optimization of [{sup 11}C]SA1 structure will be required to achieve in vivo selective GlyT-1 imaging.

Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

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Michael B Butterworth

Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC.

Science.gov (United States)

Marmor, S; Petersen, C P; Reck, F; Yang, W; Gao, N; Fisher, S L

2001-10-09

The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.

Design, synthesis and biological evaluation of uncharged catechol derivatives as selective inhibitors of PTP1B.

Science.gov (United States)

Li, Xiang-Qian; Xu, Qi; Luo, Jiao; Wang, Li-Jun; Jiang, Bo; Zhang, Ren-Shuai; Shi, Da-Yong

2017-08-18

Protein tyrosine phosphatases 1B (PTP1B) is a promising and validated therapeutic target to effectively treat T2DM and obesity. However, the development of charged PTP1B inhibitors was restricted due to their low cell permeability and poor bioavailability. Based on active natural products, two series of uncharged catechol derivatives were identified as PTP1B inhibitors by targeting a secondary aryl phosphate-binding site as well as the catalytic site. The most potent inhibitor 22 showed an IC 50 of 0.487 μM against PTP1B and strong selectivity (27-fold) over TCPTP. Kinetic studies were also performed that 22 act as a competitive PTP1B inhibitor. The treatment of C2C12 myotubes with 22 markedly increased the phosphorylation levels of IRβ, Akt and IRS1 phosphorylation. The similarity of its action profiling with that produced by insulin suggested its potential as a new non-insulin-dependent drug candidate. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Inter- and intra-specific differences in serum proteins of different species and subspecies of zebras.

Science.gov (United States)

Stratil, A; Cízová, D; Gábrisová, E; Pokorný, R

1992-11-01

1. Serum proteins of Equus grevyi, E. zebra hartmannae, E. burchelli boehmi, E. b. chapmanni and E. b. antiquorum were studied using starch-gel electrophoresis, 1-D polyacrylamide-gel electrophoresis, inhibitions of trypsin and chymotrypsin, immunoblotting, and specific staining for esterase. 2. Clear species-specific patterns were observed in albumin, transferrin, and for E. grevyi in protease inhibitor-1. Specific esterase was detected only in E. z. hartmannae. 3. Protein polymorphism was found in all studied species: E. grevyi--transferrin; E. z. hartmannae--protease inhibitor-1; E. b. boehmi--albumin, GC, transferrin, protease inhibitor-1, protease inhibitor-T; E. b. chapmanni--albumin, GC, transferrin, protease inhibitor-1; E. b. antiquorum--GC, transferrin, protease inhibitor-1. 4. Phenotype patterns of the polymorphic proteins were indicative of simple codominant inheritance. Further studies of polymorphism of protease inhibitor-2 and variability of protease inhibitor-X are needed. 5. alpha 1B glycoprotein in all zebra species was monomorphic. 6. The main transferrin components and alpha 1B glycoprotein of zebra (E. b. boehmi) were characterized for terminal sialic acid content.

Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580.

Science.gov (United States)

Conway, James G; McDonald, Brad; Parham, Janet; Keith, Barry; Rusnak, David W; Shaw, Eva; Jansen, Marilyn; Lin, Peiyuan; Payne, Alan; Crosby, Renae M; Johnson, Jennifer H; Frick, Lloyd; Lin, Min-Hwa Jasmine; Depee, Scott; Tadepalli, Sarva; Votta, Bart; James, Ian; Fuller, Karen; Chambers, Timothy J; Kull, Frederick C; Chamberlain, Stanley D; Hutchins, Jeff T

2005-11-01

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Antifungal drugs as corrosion inhibitors for aluminium in 0.1 M HCl

Energy Technology Data Exchange (ETDEWEB)

Obot, I.B. [Department of Chemistry, Faculty of Science, University of Uyo, Uyo (Nigeria)], E-mail: proffoime@yahoo.com; Obi-Egbedi, N.O. [Department of Chemistry, University of Ibadan, Ibadan (Nigeria); Umoren, S.A. [Department of Chemistry, Faculty of Science, University of Uyo, Uyo (Nigeria)

2009-08-15

The inhibitive capabilities of Clotrimazole (CTM) and Fluconazole (FLC), two antifungal drugs, on the electrochemical corrosion of aluminium in 0.1 M HCl solution has been studied using weight loss measurements at 30 and 50 deg. C. The results indicate that both compound act as inhibitors in the acidic corrodent. At constant acid concentration, the inhibition efficiency (%I) increased with increase in the concentration of the inhibitors. Increase in temperature increased the corrosion rate in the absence and presence of the inhibitors but decreased the inhibition efficiency. CTM and FLC adsorbed on the surface of aluminium according to the Langmuir adsorption isotherm model at all the concentrations and temperatures studied. Phenomenon of physical adsorption is proposed from the activation parameter obtained. Thermodynamic parameters reveal that the adsorption process is spontaneous. The reactivity of these compounds was analyzed through theoretical calculations based on AM1 semi-empirical method to explain the different efficiencies of these compounds as corrosion inhibitors. CTM was found to be a better inhibitor than FLC.

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

Science.gov (United States)

Zawadzke, Laura E; Norcia, Michael; Desbonnet, Charlene R; Wang, Hong; Freeman-Cook, Kevin; Dougherty, Thomas J

2008-02-01

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

Normalization of Hepatic Homeostasis in the Npc1nmf164 Mouse Model of Niemann-Pick Type C Disease Treated with the Histone Deacetylase Inhibitor Vorinostat.

Science.gov (United States)

Munkacsi, Andrew B; Hammond, Natalie; Schneider, Remy T; Senanayake, Dinindu S; Higaki, Katsumi; Lagutin, Kirill; Bloor, Stephen J; Ory, Daniel S; Maue, Robert A; Chen, Fannie W; Hernandez-Ono, Antonio; Dahlson, Nicole; Repa, Joyce J; Ginsberg, Henry N; Ioannou, Yiannis A; Sturley, Stephen L

2017-03-17

Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1 nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 μm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null ( Npc1 -/- ) and missense ( Npc1 nmf164 ) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Normalization of Hepatic Homeostasis in the Npc1nmf164 Mouse Model of Niemann-Pick Type C Disease Treated with the Histone Deacetylase Inhibitor Vorinostat*

Science.gov (United States)

Munkacsi, Andrew B.; Hammond, Natalie; Schneider, Remy T.; Senanayake, Dinindu S.; Higaki, Katsumi; Lagutin, Kirill; Bloor, Stephen J.; Ory, Daniel S.; Maue, Robert A.; Chen, Fannie W.; Hernandez-Ono, Antonio; Dahlson, Nicole; Repa, Joyce J.; Ginsberg, Henry N.; Ioannou, Yiannis A.; Sturley, Stephen L.

2017-01-01

Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 μm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null (Npc1−/−) and missense (Npc1nmf164) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease. PMID:28031458

Purification and properties of a novel quizalofop-p-ethyl-hydrolyzing esterase involved in quizalofop-p-ethyl degradation by Pseudomonas sp. J-2.

Science.gov (United States)

Zhang, Hui; Li, Mengya; Li, Jie; Wang, Guangli; Liu, Yuan

2017-05-10

Quizalofop-p-ethyl (QPE) is a post-emergence herbicide that effectively controls grass weeds and is often detected in the environment. However, the biochemical and molecular mechanisms of QPE degradation in the environment remains unclear. In this study, a highly effective QPE-degrading bacterial strain J-2 was isolated from acclimated activated sludge and identified as a Pseudomonas sp., containing the QPE breakdown metabolite quizalofop acid (QA) identified by Liquid Chromatography-Ion Trap-Mass Spectrometry (LC-IT-MS n ) analysis. A novel QPE hydrolase esterase-encoding gene qpeH was cloned from strain J-2 and functionally expressed in Escherichia coli BL21 (DE3). The specific activity of recombinant QpeH was 198.9 ± 2.7 U mg -1 for QPE with K m and K cat values of 41.3 ± 3.6 μM and 127.3 ± 4.5 s -1 . The optimal pH and temperature for the recombinant QpeH were 8.0 and 30 °C, respectively and the enzyme was activated by Ca 2+ , Cd 2+ , Li + , Fe 3+ and Co 2+ and inhibited by Ni 2+ , Fe 2+ , Ag + , DEPC, SDS, Tween 80, Triton X, β-mercaptoethanol, PMSF, and pCMB. In addition, the catalytic efficiency of QpeH toward different AOPP herbicides in descending order was as follows: fenoxaprop-P-ethyl > quizalofop-P-tefuryl > QPE > haloxyfop-P-methyl > cyhalofopbutyl > clodinafop-propargyl. On the basis of the phylogenetic analysis and multiple sequence alignment, the identified enzyme QpeH, was clustered with esterase family V, suggesting a new member of this family because of its low similarity of amino acid sequence with esterases reported previously.

Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

DEFF Research Database (Denmark)

Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia

2010-01-01

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and...

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

Science.gov (United States)

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG.

Science.gov (United States)

Chang, Hung-Chi; Yang, Su-Fu; Huang, Ching-Chun; Lin, Tzung-Sheng; Liang, Pi-Hui; Lin, Chun-Jung; Hsu, Lih-Ching

2013-08-01

Sodium-coupled glucose co-transporters SGLT1 and SGLT2 play important roles in intestinal absorption and renal reabsorption of glucose, respectively. Blocking SGLT2 is a novel mechanism for lowering the blood glucose level by inhibiting renal glucose reabsorption and selective SGLT2 inhibitors are under development for treatment of type 2 diabetes. Furthermore, it has been reported that perturbation of SGLT1 is associated with cardiomyopathy and cancer. Therefore, both SGLT1 and SGLT2 are potential therapeutic targets. Here we report the development of a non-radioactive cell-based method for the screening of SGLT inhibitors using COS-7 cells transiently expressing human SGLT1 (hSGLT1), CHO-K1 cells stably expressing human SGLT2 (hSGLT2), and a novel fluorescent d-glucose analogue 1-NBDG as a substrate. Our data indicate that 1-NBDG can be a good replacement for the currently used isotope-labeled SGLT substrate, (14)C-AMG. The Michaelis constant of 1-NBDG transport (0.55 mM) is similar to that of d-glucose (0.51 mM) and AMG (0.40 mM) transport through hSGLT1. The IC50 values of a SGLT inhibitor phlorizin for hSGLT1 obtained using 1-NBDG and (14)C-AMG were identical (0.11 μM) in our cell-based system. The IC50 values of dapagliflozin, a well-known selective SGLT2 inhibitor, for hSGLT2 and hSGLT1 determined using 1-NBDG were 1.86 nM and 880 nM, respectively, which are comparable to the published results obtained using (14)C-AMG. Compared to (14)C-AMG, the use of 1-NBDG is cost-effective, convenient and potentially more sensitive. Taken together, a non-radioactive system using 1-NBDG has been validated as a rapid and reliable method for the screening of SGLT1 and SGLT2 inhibitors.

Molecular population genetics of the β-esterase gene cluster of ...

Indian Academy of Sciences (India)

We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in the -esterase gene cluster. However there are some 'footprints' of directional and balancing selection shaping specific distribution of nucleotide ...

Viral kinetics in patients with chronic hepatitis C treated with the serine protease inhibitor BILN 2061

NARCIS (Netherlands)

Herrmann, Eva; Zeuzem, Stefan; Sarrazin, Christoph; Hinrichsen, Holger; Benhamou, Yves; Manns, Michael P.; Reiser, Markus; Reesink, Henk; Calleja, José L.; Forns, Xavier; Steinmann, Gerhard G.; Nehmiz, Gerhard

2006-01-01

We analysed viral kinetics from a 2-day treatment with BILN 2061, a serine protease inhibitor of hepatitis C virus, in patients chronically infected with genotype 1 hepatitis C virus. The efficiency (E), describing inhibition of viral production, was above 99.45% in all patients with minor or

Esterase isozymes patterns of grape vine (Vitis vinifera L. are altered in response to fungicide exposure

Directory of Open Access Journals (Sweden)

Gleice Ribeiro Orasmo

2015-10-01

Full Text Available Current analysis characterizes the effect of different fungicides often applied for pest control on a-and b-esterase patterns of four economically important table-wine grape cultivars (Italia, Rubi, Benitaka and Brasil of Vitis vinifera. The a- and b-esterase patterns in bud leaves of the cultivars were assessed by native PAGE analysis. Cabrio Top® compound inhibited Est-2, Est-5, Est-6, Est-7, Est-8, Est-9 and Est-10 carboxylesterases, whereas Est-4, Est-11, Est-12, Est-13, Est-14 acetylesterases and Est-16 carboxylesterase were detected as weakly stained bands. Carboxylesterases and acetylesterases were also detected as weakly stained bands when exposed to fungicides Orthocide 500®, Positron Duo® and Folicur PM®. No changes in a- and b-esterase patterns were reported when the vines were exposed to the fungicides Rovral SC®, Kumulus DF®, Curzate M®, Score® or Cuprogarb 500®. The evidence of functional changes in carboxylesterase and acetylesterase levels in current study is a warning to grape producers on the dangers inherent in the indiscriminate use of potent and modern fungicides extensively used in agriculture. The inhibition effect of fungicides on esterase isozyme molecules seems to be independent of the fungicide chemical.

A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET and Polylactic Acid (PLA

Directory of Open Access Journals (Sweden)

Georg Steinkellner

2012-02-01

Full Text Available A new esterase from Thermobifida halotolerans (Thh_Est was cloned and expressed in E. coli and investigated for surface hydrolysis of polylactic acid (PLA and polyethylene terephthalate (PET. Thh_Est is a member of the serine hydrolases superfamily containing the -GxSxG- motif with 85–87% homology to an esterase from T. alba, to an acetylxylan esterase from T. fusca and to various Thermobifida cutinases. Thh_Est hydrolyzed the PET model substrate bis(benzoyloxyethylterephthalate and PET releasing terephthalic acid and mono-(2-hydroxyethyl terephthalate in comparable amounts (19.8 and 21.5 mmol/mol of enzyme while no higher oligomers like bis-(2-hydroxyethyl terephthalate were detected. Similarly, PLA was hydrolyzed as indicated by the release of lactic acid. Enzymatic surface hydrolysis of PET and PLA led to a strong hydrophilicity increase, as quantified with a WCA decrease from 90.8° and 75.5° to 50.4° and to a complete spread of the water drop on the surface, respectively.

Molecular characterization of a novel family VIII esterase from burkholderia multivorans UWC10

CSIR Research Space (South Africa)

Rashamuse, KJ

2007-02-01

Full Text Available et al., 1992). Here we report the cloning, purification, and 3D model of a novel family VIII esterase from Burkholderia multivorans UWC10. To our knowledge no report of esterolytic activity from B. multivorans is currently available. METHODOLOGY... stream_source_info Rashamuse1_2007_d.pdf.txt stream_content_type text/plain stream_size 9884 Content-Encoding UTF-8 stream_name Rashamuse1_2007_d.pdf.txt Content-Type text/plain; charset=UTF-8 Molecular Characterization...

Carbon-11 labelling of an inhibitor of acetylcholinesterase: [[sup 11]C]physostigmine

Energy Technology Data Exchange (ETDEWEB)

Bonnot-Lours, S.; Crouzel, C.; Prenant, C.; Hinnen, F. (CEA, 91 - Orsay (France). Service Hospitalier Frederic Joliot)

1993-01-01

Physostigmine, an alkaloid from calabar bean is a strong inhibitor of acetylcholinesterase and has been used clinically in the treatment of glaucoma, atropine intoxication, myasthenia gravis and more recently, in experimental trials in Alzheimer's disease. In order to study the AChE activity in the brain by positron emission tomography, we have undertaken the labelling of physostigmine with carbon-11. The synthesis involves the reaction of [[sup 11]C]methylisocyanate with eseroline. [[sup 11]C]Methylisocyanate was obtained by heating [[sup 11]C]acetylchloride with tetrabutylammonium azide in toluene. The synthesis of [[sup 11]C]CH[sub 3]COC1 involves the carbonation of methylmagnesium bromide in THF with cyclotron produced [[sup 11]C]carbon dioxide and the addition of phthaloyl dichloride. The [[sup 11]C]methylisocyanate is distilled into a solution of eseroline in ether with a small piece of sodium. After 10 minutes at 25[sup o]C, the solution is purified by HPLC and the appropriate fraction collected. Starting with 55.5 GBq (1.5 Ci) of [[sup 11]C]carbon dioxide, 0.92-1.48 GBq (25-40 mCi) of [[sup 11]C]Physostigmine are obtained 57 minutes after EOB. (author).

Chronic effects of the ionic liquid [C4mim][Cl] towards the microalga Scenedesmus quadricauda

International Nuclear Information System (INIS)

Deng, Yun; Beadham, Ian; Wu, Jie; Chen, Xiao-Di; Hu, Lan; Gu, Jun

2015-01-01

Chronic effects of the ionic liquid [C 4 mim][Cl] (mp 73 °C) towards the microalga, Scenedesmus quadricauda were studied by flow cytometry, monitoring multiple endpoints of cell density, esterase activity, membrane integrity, reactive oxygen species and chlorophyll fluorescence. Toxicity was clearly in evidence, and although increased esterase activity indicated hormesis during initial exposure to [C 4 mim][Cl], inhibition of both esterase activity and chlorophyll fluorescence became apparent after 3 days. Cell density was also decreased by culturing with [C 4 mim][Cl], but this effect was clearly concentration-dependent and only became significant during the second half of the experiment. In contrast, [C 4 mim][Cl] had only a modest effect on reactive oxygen species (ROS) and caused little damage to cell membranes. - Highlights: • Use of an advanced biological technique, flow cytometry, to elucidate ionic liquid toxicity. • Chronic effects of ionic liquid. • Membrane integrity and ROS studied. • Mechanism of ionic liquid toxicity. - [C 4 mim][Cl] significantly inhibited esterase activity, chlorophyll fluorescence and cell density, having only a modest effect on reactive oxygen species and cell membranes

Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

Science.gov (United States)

Ostrovskyi, Dmytro; Rumpf, Tobias; Eib, Julia; Lumbroso, Alexandre; Slynko, Inna; Klaeger, Susan; Heinzlmeir, Stephanie; Forster, Michael; Gehringer, Matthias; Pfaffenrot, Ellen; Bauer, Silke Mona; Schmidtkunz, Karin; Wenzler, Sandra; Metzger, Eric; Kuster, Bernhard; Laufer, Stefan; Schüle, Roland; Sippl, Wolfgang; Breit, Bernhard; Jung, Manfred

2016-09-01

The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

Effects of high hydrostatic pressure and temperature increase on Escherichia coli spp. and pectin methyl esterase inactivation in orange juice.

Science.gov (United States)

Torres, E F; González-M, G; Klotz, B; Rodrigo, D

2016-03-01

The aim of this study was to evaluate the effect of high hydrostatic pressure treatment combined with moderate processing temperatures (25 ℃-50 ℃) on the inactivation of Escherichia coli O157: H7 (ATCC 700728), E. coli K12 (ATCC 23716), and pectin methyl esterase in orange juice, using pressures of 250 to 500 MPa with times ranging between 1 and 30 min. Loss of viability of E. coli O157:H7 increased significantly as pressure and treatment time increased, achieving a 6.5 log cycle reduction at 400 MPa for 3 min at 25 ℃ of treatment. With regard to the inactivation of pectin methyl esterase, the greatest reduction obtained was 90.05 ± 0.01% at 50 ℃ and 500 MPa of pressure for 15 min; therefore, the pectin methyl esterase enzyme was highly resistant to the treatments by high hydrostatic pressure. The results obtained in this study showed a synergistic effect between the high pressure and moderate temperatures in inactivating E. coli cells. © The Author(s) 2016.

Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

OpenAIRE

Patick, A K; Boritzki, T J; Bloom, L A

1997-01-01

Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritona...

Analyses of Evolutionary Characteristics of the Hemagglutinin-Esterase Gene of Influenza C Virus during a Period of 68 Years Reveals Evolutionary Patterns Different from Influenza A and B Viruses

Directory of Open Access Journals (Sweden)

Yuki Furuse

2016-11-01

Full Text Available Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1 multiple lineages have been circulating globally; (2 there have been weak and infrequent selective bottlenecks; (3 the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4 there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics.

Electrochemical and quantum chemical studies of some indole derivatives as corrosion inhibitors for C38 steel in molar hydrochloric acid

International Nuclear Information System (INIS)

Lebrini, M.; Robert, F.; Vezin, H.; Roos, C.

2010-01-01

A comparative study of 9H-pyrido[3,4-b]indole (norharmane) and 1-methyl-9H-pyrido[3,4-b]indole (harmane) as inhibitors for C38 steel corrosion in 1 M HCl solution at 25 o C was carried out. Potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) techniques were applied to study the metal corrosion behavior in the absence and presence of different concentrations of these inhibitors. The OCP as a function of time were also established. Cathodic and anodic polarization curves show that norharmane and harmane are a mixed-type inhibitors. Adsorption of indole derivatives on the C38 steel surface, in 1 M HCl solution, follows the Langmuir adsorption isotherm model. The ΔG ads o values were calculated and discussed. The potential of zero charge (PZC) of the C38 steel in inhibited solution was studied by the EIS method, and a mechanism for the adsorption process was proposed. Raman spectroscopy confirmed that indole molecules strongly adsorbed onto the steel surface. The electronic properties of indole derivates, obtained using the AM1 semi-empirical quantum chemical approach, were correlated with their experimental efficiencies using the linear resistance model (LR).

Inhibitor scaffold for the histone lysine demethylase KDM4C (JMJD2C)

DEFF Research Database (Denmark)

Leurs, Ulrike; Clausen, Rasmus P; Kristensen, Jesper L

2012-01-01

The human histone demethylases of the KDM4 (JMJD2) family have been associated to diseases such as prostate and breast cancer, as well as X-linked mental retardation. Therefore, these enzymes are considered oncogenes and their selective inhibition might be a possible therapeutic approach to treat...... cancer. Here we describe a heterocyclic ring system library screened against the histone demethylase KDM4C (JMJD2C) in the search for novel inhibitory scaffolds. A 4-hydroxypyrazole scaffold was identified as an inhibitor of KDM4C; this scaffold could be employed in the further development of novel...... therapeutics, as well as for the elucidation of the biological roles of KDM4C on epigenetic regulation....

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

Directory of Open Access Journals (Sweden)

Anna Figueiredo

2006-11-01

Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs target HIV-1 reverse transcriptase (RT by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

NARCIS (Netherlands)

M. Pieters (Marlien); S.A. Barnard (Sunelle A.); D.T. Loots (Du Toit); D.C. Rijken (Dingeman)

2017-01-01

textabstractDue to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen

Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots.

Science.gov (United States)

Negrel, Jonathan; Javelle, Francine; Morandi, Dominique; Lucchi, Géraldine

2016-12-01

A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K m  = 2 μM) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere. Copyright © 2016 Elsevier Masson SAS. All rights

C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

Science.gov (United States)

Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

2015-09-11

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[sup 14]C-labeling of a tetrahydroacridine, a novel CNS-selective cholinesterase inhibitor

Energy Technology Data Exchange (ETDEWEB)

Nishioka, Kazuhiko; Kamada, Takeshi; Kanamaru, Hiroshi (Sumitomo Chemical Co., Ltd., Takatsukasa, Takarazuka (Japan). Environmental Health Science Lab.)

1992-06-01

9-Amino-8-fluoro-2,4-methano-1,2,3,4-tetrahydroacridine citrate (SM-10888), a novel cholinesterase inhibitor, was labeled with carbon-14 at C9 of the tetrahydroacridine ring for use in metabolic studies. Carbonation of 2,6-difluorophenyllithium (3) with [[sup 14]C]carbon dioxide gave the acid (4). Chlorination of 4 followed by treatment of the resulting acid chloride with ammonia afforded the amide (5). Dehydration of 5 with thionyl chloride and subsequent displacement reaction with ammonia gave the aminobenzonitrile (7). Condensation of 7 with the ketone (8) in the presence of anhydrous zinc chloride yielded the aminoacridine (9), which was treated with citric acid to afford [9-[sup 14]C]SM-10888 (1). The overall yield of 1 was 37% from 2, and the specific activity was 1.35 GBq/mmol. (author).

A Thermally Stable Form of Bacterial Cocaine Esterase: A Potential Therapeutic Agent for Treatment of Cocaine Abuse

Energy Technology Data Exchange (ETDEWEB)

Brim, Remy L.; Nance, Mark R.; Youngstrom, Daniel W.; Narasimhan, Diwahar; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K.; Woods, James H. (Michigan); (Michigan-Med); (Kentucky)

2010-09-03

Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-{angstrom} resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.

N- and C-alkylation of seven-membered iminosugars generates potent glucocerebrosidase inhibitors and F508del-CFTR correctors.

Science.gov (United States)

Désiré, J; Mondon, M; Fontelle, N; Nakagawa, S; Hirokami, Y; Adachi, I; Iwaki, R; Fleet, G W J; Alonzi, D S; Twigg, G; Butters, T D; Bertrand, J; Cendret, V; Becq, F; Norez, C; Marrot, J; Kato, A; Blériot, Y

2014-11-28

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a β-glucosidase and β-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent β-glucosidase, β-galactosidase, α-L-rhamnosidase and β-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 μM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.

Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

Energy Technology Data Exchange (ETDEWEB)

Risveden, Klas; Bhand, Sunil; Danielsson, Bengt [Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund (Sweden); Dick, Kimberly A; Samuelson, Lars [Solid State Physics, Lund University, Box 118, S-22100 Lund (Sweden); Rydberg, Patrik, E-mail: Kimberly.Dick@ftf.lth.se [Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)

2010-02-05

A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN{sub x}-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area and thereby higher enzyme binding capacity. A theoretical calculation is included to show how the enzyme kinetics and hence the sensitivity can be influenced and increased by the control of electrical fields in relation to the active sites of enzymes in an electronic biosensor. The possible effects of electrical fields employed in the RISFET on the function of acetylcholine esterase is investigated using quantum chemical methods, which show that the small electric field strengths used are unlikely to affect enzyme kinetics. Acetylcholine esterase activity is determined using choline oxidase and peroxidase by measuring the amount of choline formed using the chemiluminescent luminol reaction.

The Alpha-Defensin Immunoassay and Leukocyte Esterase Colorimetric Strip Test for the Diagnosis of Periprosthetic Infection

Science.gov (United States)

Wyatt, M.C.; Beswick, A.D.; Kunutsor, S.K.; Wilson, M.J.; Whitehouse, M.R.; Blom, A.W.

2016-01-01

Background: Synovial biomarkers have recently been adopted as diagnostic tools for periprosthetic joint infection (PJI), but their utility is uncertain. The purpose of this systematic review and meta-analysis was to synthesize the evidence on the accuracy of the alpha-defensin immunoassay and leukocyte esterase colorimetric strip test for the diagnosis of PJI compared with the Musculoskeletal Infection Society diagnostic criteria. Methods: We performed a systematic review to identify diagnostic technique studies evaluating the accuracy of alpha-defensin or leukocyte esterase in the diagnosis of PJI. MEDLINE and Embase on Ovid, ACM, ADS, arXiv, CERN DS (Conseil Européen pour la Recherche Nucléaire Document Server), CrossRef DOI (Digital Object Identifier), DBLP (Digital Bibliography & Library Project), Espacenet, Google Scholar, Gutenberg, HighWire, IEEE Xplore (Institute of Electrical and Electronics Engineers digital library), INSPIRE, JSTOR (Journal Storage), OAlster (Open Archives Initiative Protocol for Metadata Harvesting), Open Content, Pubget, PubMed, and Web of Science were searched for appropriate studies indexed from inception until May 30, 2015, along with unpublished or gray literature. The classification of studies and data extraction were performed independently by 2 reviewers. Data extraction permitted meta-analysis of sensitivity and specificity with construction of receiver operating characteristic curves for each test. Results: We included 11 eligible studies. The pooled diagnostic sensitivity and specificity of alpha-defensin (6 studies) for PJI were 1.00 (95% confidence interval [CI], 0.82 to 1.00) and 0.96 (95% CI, 0.89 to 0.99), respectively. The area under the curve (AUC) for alpha-defensin and PJI was 0.99 (95% CI, 0.98 to 1.00). The pooled diagnostic sensitivity and specificity of leukocyte esterase (5 studies) for PJI were 0.81 (95% CI, 0.49 to 0.95) and 0.97 (95% CI, 0.82 to 0.99), respectively. The AUC for leukocyte esterase and PJI

Molecular characterization of c-Abl/c-Src kinase inhibitors targeted against murine tumour progenitor cells that express stem cell markers.

Directory of Open Access Journals (Sweden)

Thomas Kruewel

Full Text Available BACKGROUND: The non-receptor tyrosine kinases c-Abl and c-Src are overexpressed in various solid human tumours. Inhibition of their hyperactivity represents a molecular rationale in the combat of cancerous diseases. Here we examined the effects of a new family of pyrazolo [3,4-d] pyrimidines on a panel of 11 different murine lung tumour progenitor cell lines, that express stem cell markers, as well as on the human lung adenocarcinoma cell line A549, the human hepatoma cell line HepG2 and the human colon cancer cell line CaCo2 to obtain insight into the mode of action of these experimental drugs. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with the dual kinase inhibitors blocked c-Abl and c-Src kinase activity efficiently in the nanomolar range, induced apoptosis, reduced cell viability and caused cell cycle arrest predominantly at G0/G1 phase while western blot analysis confirmed repressed protein expression of c-Abl and c-Src as well as the interacting partners p38 mitogen activated protein kinase, heterogenous ribonucleoprotein K, cyclin dependent kinase 1 and further proteins that are crucial for tumour progression. Importantly, a significant repression of the epidermal growth factor receptor was observed while whole genome gene expression analysis evidenced regulation of many cell cycle regulated genes as well integrin and focal adhesion kinase (FAK signalling to impact cytoskeleton dynamics, migration, invasion and metastasis. CONCLUSIONS/SIGNIFICANCE: Our experiments and recently published in vivo engraftment studies with various tumour cell lines revealed the dual kinase inhibitors to be efficient in their antitumour activity.

Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

Directory of Open Access Journals (Sweden)

Chunzi Liang

2014-01-01

Full Text Available Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB, a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM, alanine (0.5 mM, valine (0.5 mM, EX527 (SIRT1 inhibitor, 25 μM, and Compound C (AMPK inhibitor, 25 μM alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.

1H and 13C NMR Chemical Shift Assignments and Conformational Analysis for the Two Diastereomers of the Vitamin K Epoxide Reductase Inhibitor Brodifacoum

International Nuclear Information System (INIS)

Cort, John R.; Cho, Herman M.

2009-01-01

Proton and 13C NMR chemical shift assignments and 1H-1H scalar couplings for the two diastereomers of the vitamin K epoxide reductase (VKOR) inhibitor brodifacoum have been determined from acetone solutions containing both diastereomers. Data were obtained from homo- and heteronuclear correlation spectra acquired at 1H frequencies of 750 and 900 MHz over a 268-303 K temperature range. Conformations inferred from scalar coupling and 1-D NOE measurements exhibit large differences between the diastereomers. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

Synthesis activity-based zymography for detection of lipases and esterases.

Science.gov (United States)

Kwon, Min-A; Kim, Hyun Suk; Hahm, Dae-Hyun; Song, Jae Kwang

2011-04-01

A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.

PD-1/PD-L1 Inhibitors for Immuno-oncology: From Antibodies to Small Molecules.

Science.gov (United States)

Geng, Qiaohong; Jiao, Peifu; Jin, Peng; Su, Gaoxing; Dong, Jinlong; Yan, Bing

2018-02-12

The recent regulatory approvals of immune checkpoint protein inhibitors, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab ushered a new era in cancer therapy. These inhibitors do not attack tumor cells directly but instead mobilize the immune system to re-recognize and eradicate tumors, which endows them with unique advantages including durable clinical responses and substantial clinical benefits. PD-1/PD-L1 inhibitors, a pillar of immune checkpoint protein inhibitors, have demonstrated unprecedented clinical efficacy in more than 20 cancer types. Besides monoclonal antibodies, diverse PD- 1/PD-L1 inhibiting candidates, such as peptides, small molecules have formed a powerful collection of weapons to fight cancer. The goal of this review is to summarize and discuss the current PD-1/PD-L1 inhibitors including candidates under clinical development, their molecular interactions with PD-1 or PD-L1, the disclosed structureactivity relationships of peptides and small molecules as inhibitors. Current PD-1/PD-L1 inhibitors under clinical development are exclusively dominated by antibodies. The molecular interactions of therapeutic antibodies with PD-1 or PD-L1 have been gradually elucidated for the design of novel inhibitors. Various peptides and traditional small molecules have been investigated in preclinical model to discover novel PD-1/PD-L1 inhibitors. Peptides and small molecules may play an important role in immuno-oncology because they may bind to multiple immune checkpoint proteins via rational design, opening opportunity for a new generation of novel PD-1/PD-L1 inhibitors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Novel selective PDE type 1 inhibitors cause vasodilatation and lower blood pressure in rats

DEFF Research Database (Denmark)

Laursen, Morten; Beck, Lilliana; Kehler, Jan

2017-01-01

BACKGROUND AND PURPOSE: The PDE enzymes (PDE1-11) hydrolyse and thus inactivate cyclic nucleotides and are important in the regulation of the cardiovascular system. Here,we have investigated the effects on the cardiovascular system, of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027...... and Lu AF58027 inhibited PDE1A, PDE1B and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT-PCR revealed expression of PDE1A, PDE1B and PDE1C in rat brain, heart and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated...... and Lu AF58027 dose-dependently lowered mean BP and increased heart rate. In conscious rats with telemetric pressure transducers, repeated dosing with Lu AF41228 lowered mean arterial BP 10-15 mmHg and increased heart rate. CONCLUSIONS AND IMPLICATIONS: These novel PDE1 inhibitors induce vasodilation...

A chiral synthesis of dapoxetine hydrochloride, a serotonin re-uptake inhibitor, and its 14C isotopomer

International Nuclear Information System (INIS)

Wheeler, W.J.; O'Bannon, D.D.

1992-01-01

The 14 C-isotopmer of dapoxetine-[ 14 C] HCl (S (+) -N,N-dimethyl-α[2-(1-naphthalenyloxy)ethyl-2- 14 C]benzenemeth a-n amine hydrochloride, 1a), a potent serotonin re-uptake inhibitor has been prepared by a chiral synthesis, starting with tert. -butyloxyphenylglycine (3). Borane reduction, followed by activation of the resulting alcohol 4 as its mesylate 5b, provided the chiral starting material. The radiolabel was introduced by reaction of 5b with sodium cyanide-[ 14 C]. The desired product (1) was then elaborated from nitrile 6a,b via a five step synthesis in an overall 19.5% radiochemical yield. (Author)

Different effects of histone deacetylase inhibitors nicotinamide and trichostatin A (TSA) in C17.2 neural stem cells.

Science.gov (United States)

Wang, Haifeng; Cheng, Hua; Wang, Kai; Wen, Tieqiao

2012-11-01

Histone deacetylase inhibitors are involved in proliferation, apoptosis, cell cycle, mRNA transcription, and protein expression in various cells. However, the molecular mechanism underlying such functions is still not fully clear. In this study, we used C17.2 neural stem cell (NSC) line as a model to evaluate the effects of nicotinamide and trichostatin A (TSA) on cell characteristics. Results show that nicotinamide and TSA greatly inhibit cell growth, lead to cell morphology changes, and effectively induce cell apoptosis in a dose-dependent manner. Western blot analyses confirmed that nicotinamide significantly decreases the expression of bcl-2 and p38. Further insight into the molecular mechanisms shows the suppression of phosphorylation in eukaryotic initiation factor 4E-binding protein 1 (4EBP1) by nicotinamide, whereas, an increased expression of bcl-2 and p38 and phosphorylation of 4EBP1 by TSA. However, both nicotinamide and TSA significantly increase the expression of cytochrome c (cyt c). These results strongly suggest that bcl-2, p38, cyt c, and p-4EBP1 could suppress proliferation and induce apoptosis of C17.2 NSCs mediated by histone deacetylase inhibitors, nicotinamide and TSA, involving different molecular mechanisms.