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Enzyme Substrate (enzyme + substrate)
Selected AbstractsEvaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococciJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2006J.D. Perry Abstract Aims:, Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of , -glucuronidase and , -glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. Methods and Results:, A novel , -glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl- , - d -glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four , -glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl- , - d -glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised , -glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid- , - d -glucoside was inferior. However, the variability in detectable , -glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. Conclusions:, The , -glucuronidase substrate CMUG appears to be a more promising detection system than the various , -glucosidase substrates tested. Significance and Impact of the Study:, The novel substrate CMUG showed enhanced sensitivity for the detection of , -glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples. [source] The Role of Axial Ligation in Nitrate Reductase: A Model Study by DFT Calculations on the Mechanism of Nitrate ReductionEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2008Kuntal Pal Abstract The reactivity differences of the model anionic complexes [Mo(mnt)2(X)(PPh3)], [mnt2, = 1,2-dicyanoethylenedithiolate; X = SPh (1a), SEt (1b), Cl (1c), Br (1b)] towards oxygen atom transfer from nitrate, which is a key step performed by nitrate reductase, has been investigated by density functional theory calculations. Unlike complexes 1a and 1b, complexes 1c and 1d do not react with nitrate. Thermodynamically, all these complexes have a similar ability to generate the pentacoordinate active state [Mo(mnt)2(X)], by dissociation of PPh3, although the inaccessibility of the dxy orbital in 1c,d and the instability of the corresponding nitrate-bound enzyme substrate (ES) type complex contributes to their failure to reduce nitrate. The nature of the ES complex for 1a,b is described. The variation in the experimental data due to the change of axial ligation from SPh to SEt on the catalytic pathway has also been addressed. The gas-phase and solvent-corrected potential energy surface for the reaction of 1a,b with nitrate are established with fully optimized minima and transition states.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sproutsJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2003K. Warriner Abstract Aims: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. Methods and Results:E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20 000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ , -glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. Conclusions:E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. Significance and Impact of the Study: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts. [source] A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking waterJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2000S.O. Van Poucke A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of ,-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-,- d -glucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated ,,90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5·4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for ,emergency' monitoring of drinking water when rapid results are crucial. [source] Self-immolative dendrimers as novel drug delivery platformsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 5 2006Doron Shabat Abstract Self-immolative dendrimers were recently developed and introduced as a potential platform for a single-triggered multi-prodrug. These unique structural dendrimers can release all of their tail units through domino-like chain fragmentation, which is initiated by a single cleavage at the dendrimer core. The incorporation of drug molecules as the tail units and an enzyme substrate as the trigger generates a multi-prodrug unit that is activated with a single enzymatic cleavage. We have demonstrated several examples of self-immolative dendritic prodrug systems and have shown significant advantages with respect to the appropriate monomeric prodrug. We anticipate that single-triggered, dendritic prodrugs will be exploited to further improve selective chemotherapeutic approaches in cancer therapy. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1569,1578, 2006 [source] Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of ,7 nicotinic receptors and internalization of NMDA receptorsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2010H Shen BACKGROUND AND PURPOSE Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil. EXPERIMENTAL APPROACH Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca2+ concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD. KEY RESULTS Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca2+ entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of ,7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca2+ entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca2+ entry induced by donepezil. CONCLUSIONS AND IMPLICATIONS Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of ,7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system. [source] Probing the Activity of Matrix Metalloproteinase II with a Sequentially Click-Labeled Silica Nanoparticle FRET ProbeCHEMBIOCHEM, Issue 14 2009Daniela E. Achatz Selective double click labeling: The determination of nanomolar concentrations of matrix metalloproteinase II is made possible through the use of a nanoprobe that consists of fluorescently doped silica nanoparticles to which a FRET-based enzyme substrate has been conjugated through sequential click reactions. [source] Direct enantioselective HPLC monitoring of lipase-catalyzed kinetic resolution of tiaprofenic acid in nonstandard HPLC organic solvents,CHIRALITY, Issue 8 2008Ashraf Ghanem Abstract The first straightforward lipase-catalyzed enantioselective access to enantiomerically enriched tiaprofenic acid as a versatile method in chiral separation of racemates is demonstrated. The latter was directly monitored by enantioselective HPLC using a 3,5-dimethylphenylcarbamate derivative of cellulose-based chiral stationary phase namely Chiralpak IB (the immobilized version of Chiralcel OD). Non-standard HPLC organic solvents were used as diluent to dissolve the "difficult to dissolve" enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. The existence of a non-standard HPLC organic solvent (e.g., methyl tert -butyl ether) in the mobile phase composition is mandatory to accomplish the simultaneous enantioselective HPLC baseline separation of both substrate and product. Chirality, 2008. © 2008 Wiley-Liss, Inc. [source] Polydivinylbenzene/Ethylvinylbenzene Composite Membranes for the Optimization of a Whole Blood Glucose SensorELECTROANALYSIS, Issue 1 2006Kerry Bridge Abstract A novel ultra thin polydivinylbenzene/ethylvinylbenzene composite membrane has been developed for use as the outer covering barrier in a model amperometric glucose oxidase enzyme electrode. The composite membrane was formed via the cathodic electropolymerization of divinylbenzene/ethylvinylbenzene at the surface of gold sputter coated host alumina membranes, (serving solely as a mechanical support for the thin polymer film). Permeability coefficients were determined for the enzyme substrates, O2 and glucose, across composite membranes formed with a range of polymer thicknesses. Due to the highly substrate diffusion limiting nature of the composite membrane, it was found that anionic interferents present in blood (such as ascorbate), were effectively screened from the working electrode via a charge exclusion mechanism, in a manner similar to previous findings within our laboratory. The enzyme electrode showed an initial 32% signal drift when first exposed to whole human blood over a period of 2 hours, after which time enzyme electrode responses remained essentially stable. Whole blood patient glucose determinations yielded a correlation coefficient of r2=0.97 in comparison to standard hospital analyses. [source] Real-Time Liquid Crystal pH Sensor for Monitoring Enzymatic Activities of PenicillinaseADVANCED FUNCTIONAL MATERIALS, Issue 23 2009Xinyan Bi Abstract A liquid crystal (LC)-based pH sensor for real-time monitoring of changes in localized pH values near a solid surface is reported, along with its application for the detection of enzymatic activities. It is found that 4-cyano-4,-pentylbiphenyl (5CB), when doped with 4,-pentyl-biphenyl-4-carboxylic acid (PBA), shows a bright-to-dark optical response to a very small change in pH (from 6.9 to 7.0). The pH-driven optical response can be explained by using orientational transitions of 5CB induced by the protonation and deprotonation of PBA at the aqueous/LC interface. Because of its high pH sensitivity, the LC-based sensor is further exploited for monitoring local pH changes resulting from enzymatic reactions. As a proof of concept, the hydrolysis of penicillin G by surface-immobilized penicillinase is monitored using the system, even when the concentration of penicillin G is as low as 1,nM. This type of LC-based sensor may find potential utilities in high-throughput screening of enzyme substrates and enzyme inhibitors. [source] The application of chromogenic media in clinical microbiologyJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007J.D. Perry Summary Since 1990, a wide range of chromogenic culture media has been made commercially available providing useful tools for diagnostic clinical microbiology. By the inclusion of chromogenic enzyme substrates targeting microbial enzymes, such media are able to target pathogens with high specificity. Examples of target pathogens include Staphylococcus aureus, Streptococcus agalactiae, Salmonella spp. and Candida spp. The inclusion of multiple chromogenic substrates into culture media facilitates the differentiation of polymicrobial cultures, thus allowing for the development of improved media for diagnosis of urinary tract infections and media for the enhanced discrimination of yeasts. The purpose of this review is to provide some insight into how such media work and appraise their utility in routine clinical diagnostics, in comparison with conventional media. [source] Activity-based mass spectrometric characterization of proteases and inhibitors in human salivaPROTEOMICS - CLINICAL APPLICATIONS, Issue 7 2009Xiuli Sun Abstract Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50,75,kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer. [source] Novel structural features in the GMC family of oxidoreductases revealed by the crystal structure of fungal aryl-alcohol oxidaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Israel S. Fernández Lignin biodegradation, a key step in carbon recycling in land ecosystems, is carried out by white-rot fungi through an H2O2 -dependent process defined as enzymatic combustion. Pleurotus eryngii is a selective lignin-degrading fungus that produces H2O2 during redox cycling of p -anisylic compounds involving the secreted flavoenzyme aryl-alcohol oxidase (AAO). Here, the 2.4,Ĺ resolution X-ray crystal structure of this oxidoreductase, which catalyzes dehydrogenation reactions on various primary polyunsaturated alcohols, yielding the corresponding aldehydes, is reported. The AAO crystal structure was solved by single-wavelength anomalous diffraction of a selenomethionine derivative obtained by Escherichia coli expression and in vitro folding. This monomeric enzyme is composed of two domains, the overall folding of which places it into the GMC (glucose,methanol,choline oxidase) oxidoreductase family, and a noncovalently bound FAD cofactor. However, two additional structural elements exist in the surroundings of its active site that modulate the access of substrates; these are absent in the structure of the model GMC oxidoreductase glucose oxidase. The folding of these novel elements gives rise to a funnel-like hydrophobic channel that connects the solvent region to the buried active-site cavity of AAO. This putative active-site cavity is located in front of the re side of the FAD isoalloxazine ring and near two histidines (His502 and His546) that could contribute to alcohol activation as catalytic bases. Moreover, three aromatic side chains from two phenylalanines (Phe397 and Phe502) and one tyrosine (Tyr92) at the inner region of the channel form an aromatic gate that may regulate the access of the enzyme substrates to the active site as well as contribute to the recognition of the alcohols that can effectively be oxidized by AAO. [source] Latent Blue and Red Fluorophores Based on the Trimethyl LockCHEMBIOCHEM, Issue 8 2006Luke D. Lavis Unlocking fluorescence. The "trimethyl lock" is an effective way to mask structurally diverse fluorescent molecules for the preparation of fluorogenic enzyme substrates. These novel probes exhibit remarkable stability in water, and this makes them useful for a variety of biochemical and biological applications. [source] Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein PrenylationCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2008Amanda J. DeGraw Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications. [source] | ||||||||||||||||