Ellman's Reagent Research Articles

Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE.

Genotoxic Effects of Carotenoid Breakdown Products in Human Retinal Pigment Epithelial Cells

Purpose: To investigate the genotoxic effects of lutein (LBP) and β -carotene breakdown products (β -apo-8-carotenal, BA8C) and the preventive role of GSH in human retinal pigment epithelial cells (ARPE-19). Methods: LBP- and BA8C-induced DNA damage in human retinal pigment epithelial cells (ARPE-19) was determined by comet assay. The DNA damage was quantified by the image analysis system using Comet Score™ software. ARPE-19 cell viability was determined by CellTiter 96 AQueous one-solution cell proliferation assay kit. Intracellular GSH levels were measured by Ellman's reagent. Results: Incubation of serum-starved ARPE-19 cells with LBP and BA8C caused significant DNA damage in a dose- and time-dependent manner. The DNA damage and cell death incurred by LBP and BA8C were significantly prevented by N-acetylcysteine (NAC) but not by α -tocopherol + ascorbic acid (T + AA). Furthermore, BSO-induced GSH depletion in ARPE-19 cells caused a significant elevation in LBP- and BA8C-induced DNA damage, whereas increased GSH levels in ARPE-19 cells prevented it. Conclusions: Our results suggest that breakdown products of dietary carotenoids could be genotoxic in ARPE-19 cells. LBP-induced genotoxic effects could worsen oxidative stress. The intracellular GSH pool in ARPE-19 cells might play a critical role in carotenoid breakdown products-induced genotoxicity.

<I>Triclisia sacleuxii</I> (Pierre) Diels (Menispermaceae), a potential source of acetylcholinesterase inhibitors

To search for compounds possibly useful for the treatment of Alzheimer's disease. Alkaloid fractions from the roots, stems and leaves of Triclisia sacleuxii (Menispermaceae) and pure bisbenzylisoquinoline alkaloids isolated from the roots (phaeanthine, N-methylapateline, 1,2-dehydroapateline and gasabiimine) were assessed for acetylcholinesterase inhibitory activity. All extracts and compounds tested inhibited acetylcholinesterase to varying degrees; the leaf tertiary alkaloid fractions and the root quaternary alkaloid fractions exhibited the strongest inhibitory potential (90% at 0.1 mg/ml). The leaf tertiary alkaloid fraction was selected for further analysis (the quaternary alkaloids, which are too polar for absorption and brain distribution, were presumed to be clinically uninteresting). TLC bioautography using Ellman's reagent allowed the detection of acetylcholinesterase inhibitors and the isolation of the major active constituent, which was identified as lindoldhamine, a one-bridged bisbenzylisoquinoline alkaloid. Lindoldhamine displayed high acetylcholinesterase inhibitory activity with a 50% inhibition concentration in the micromolar range. All T. sacleuxii alkaloid fractions tested exhibited anti-acetylcholinesterase activity; isolated bisbenzylisoquinoline alkaloids showed weak-to-high inhibition depending on their structural features. Structure modification could provide interesting derivatives with enhanced anti-acetylcholinesterase activity.

Leishmanicidal metabolites from Cochliobolus sp., an endophytic fungus isolated from Piptadenia adiantoides (Fabaceae).

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman's reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL−1. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC50 values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.

Increased Acetylcholinesterase Activity of Microparticles Derived from Red Cells (RMP) Compared to Platelets (PMP).

INTRODUCTION. Cell-derived microparticles (MP) such as from platelets (PMP), endothelia (EMP) and leukocytes (LMP) are increasingly recognized as useful biomarker and important mediators of thrombosis and inflammation. However, little attention has been paid to the possible role of MP from RBC (RMP) in vascular disorders. RMP were identified by glycophorin (GPH) in flow cytometry in most studies. We reported heterogeneity of RMP in size and phenotypes and that GPH is expressed predominately in larger RMP, not in smaller RMP and that GPH+ RMP are more active than GPH- RMP in thrombin generation. Since acetylcholinesterase (AChE) activity has been measured on RMP, and was recently proposed as a marker of some inflammatory states, we investigated AChE activity of RMP compared to platelet-derived MP (PMP). AChE of PMP has not previously been reported.METHODS. RMP were prepared from intact washed RBC at 18% Ht exposed to calcium ionophore (4uM) in presence of calcium (2mM) for 30 min. PMP were prepared from 20 mL citrated blood, and exposing the platelet-rich plasma to 1 uM calcium ionophore (without added Ca2+) and collagen, 4ug/mL, for 20 min. AChE assay was based on Ellman's method and reagent (DTNB), run in 96-well plates, 300uL. Substrate was acetylthiocholine iodide (1 mM f.c.). DTNB was used at 0.67 mM f.c. Tests were run +/− quinidine (Q) (1.2 uM) and some tests were in presence of saponin 0.01%. Q is known to inhibit AChE of plasma but RBC activity is insensitive. Activity is expressed in umols substrate cleaved /min per 108 MP, with provisos below. Flow cytometry using FITC labeled lectin, Ulex europaeus (Ulex) was used to quantitate RMP and PMP.RESULTS.As expected, Q inhibited AChE in plasma by >90% but not AChE of RMP. On contrary, RMP were consistently stimulated by Q, up to 150% activity +Q; some preparations of PMP were also stimulated.Saponin, which has been used in assay of RBC AChE, had little effect on PMP or RMP activity.In 12 experiments, AChE of PMP exhibited marked concentration-dependence. The apparent activity per mL of suspension was greater with lesser volumes, by as much as 3-fold between 2.5uL and 20uL added. This could not be explained by substrate inhibition since the effect varied in different preparations, was absent in particle-free plasma, and did not diminish in low substrate. This suggests the presence of a natural inhibitor.Calculation of specific activity of the MP was complicated by the dependence of apparent activity on volume assayed. However, when equal dilutions were compared, a representative experiment showed RMP had about 6-fold greater activity than PMP per 108 MP: 36.0 vs. 5.88 for 2.5uL suspension; and 29.0 vs. 3.9 for 20 uL assayed, in units above.CONCLUSIONS / DISCUSSION. The AChE activity of RMP is about 6-fold greater than PMP. Weaker activity on PMP is possibly attributed to a previously unreported natural inhibitor. Blood AChE activity has been shown to reflect inflammatory states. Since AChE is a GPI-anchored protein, it is preferentially depleted from cells on the MP shed off. Assay of this activity in patient cell-free plasma, +/− Q, may be a useful biomarker. It is well known that hemolytic anemia, where RMP are elevated, is often associated with thrombotic complications, whereas ITP, where PMP are frequently elevated, rarely is. Further study to characterize AChE in RMP and other MP, and to clarify the physiological role of MP- and cell-associated AChE in thrombosis, inflammation, and cardiovascular disease is in progress.

Covalent immobilization of urease on polysiloxane matrices containing 3-aminopropyl and 3-mercaptopropyl groups

A technique of covalent immobilization of urease on polysiloxane templates, involving the sol-gel method, based on the use of crosslinking reagents (such as glutaraldehyde and Ellman's reagent) has been proposed. Urease, covalently grafted onto the surface of a poly(3-mercaptopropyl)siloxane template, was shown to retain its activity (67-84%) and stability (a decrease of 10% was observed over a period of 300 days). Urease adsorbed onto the poly(3-mercaptopropyl)siloxane template exhibited a higher activity than the native enzyme. The 3-mercaptopropyl groups of the polysiloxane template could be brought into the vicinity of the active metal center of the adsorbed urease and start acting as proton donors, thereby increasing the rate of the reaction catalyzed by the enzyme. Covalent immobilization of urease onto a 3-aminopropyl-containing polysiloxane template was shown to be less efficient, because it resulted in considerable losses of the activity of the enzyme. Conversely, urease adsorbed onto this template exhibited a high activity (60-86%).

Changes in Erythrocyte Glutathione and Plasma Membrane Calcium Pump in Preterm Newborns Treated Antenatally with MgSO4

Background: Magnesium is required for the proper activity of many metabolic pathways in every cell type. Mg deficiency gives rise to preterm birth and low body weight that are associated with pathological circumstances, including disturbed ions homeostasis and insufficient antioxidant protection. Antenatal MgSO₄ treatment has been reported to exhibit a protective effect on the developing fetus; however, the molecular mechanism of this protection remains not fully understood. Objectives: Since Mg ions very rapid cross the placenta, our study tested the hypothesis whether prenatal exposure to MgSO₄ may modify the expression and activity of erythrocyte plasma membrane calcium pump (PMCA), and total erythrocyte glutathione (GSH) concentration in preterm newborns. Methods: Immunocharacteristics of erythrocyte PMCA in control and MgSO₄-treated newborns were done using general and isoform-specific antibodies. The hydrolytic activity of PMCA was determined with and without calmodulin. Total GSH erythrocyte levels were analyzed by a quantitative assay using Ellman reagent. All assays were done after delivery and 24 h later. Results: Significant differences were present in PMCA amount, isoform composition, basal activity and sensitivity for stimulation by calmodulin in both groups of neonates. The total GSH content altered during examined time, but only in the control group. Conclusions: Prenatal treatment with MgSO₄ may exert a significant influence on calcium homeostasis and nonenzymatic antioxidant reserve in the erythrocytes of preterm newborns.

A 1,536-Well-Based Kinetic HTS Assay for Inhibitors ofSchistosoma mansoniThioredoxin Glutathione Reductase

Abstract: Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.

Improvement of acetylcholinesterase-based assay for organophosphates in way of identification by reactivators

Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellman's reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.

Isolated Cytochrome c Oxidase Deficiency in G93A SOD1 Mice Overexpressing CCS Protein

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Hyaluronan Constitutively Regulates Activation of COX-2-mediated Cell Survival Activity in Intestinal Epithelial and Colon Carcinoma Cells

Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, beta-catenin, and cyclooxygenase-2/prostaglandin E(2) in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 --> phosphoinositide 3-kinase/AKT --> beta-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E(2)-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer.

Determination of Sulfite in Wine by Linear Sweep Voltammetry

AbstractA new sensitive electrochemical assay of free sulfite in wine samples at a bare glassy carbon electrode is reported. The method is based on the titration of sulfite with Ellman's reagent using voltammetric detection of the final point. The electrochemical sensing protocol should facilitate rapid field screening of sulfite preservatives.

Identical kinetics of human erythrocyte and muscle acetylcholinesterase with respect to carbamate pre-treatment, residual activity upon soman challenge and spontaneous reactivation after withdrawal of the inhibitors

The efficacy of oxime treatment in soman poisoning is limited due to rapid aging of inhibited acetylcholinesterase (AChE). Pre-treatment with carbamates was shown to improve antidotal treatment substantially. Recently, by using a dynamically working in vitro model with real-time determination of membrane-bound AChE activity, we were able to demonstrate that pre-inhibition of human erythrocyte AChE with pyridostigmine or physostigmine resulted in a markedly higher residual AChE activity after inhibition by soman or paraoxon than in the absence of reversible inhibitors. The purpose of the present study was to compare the effect of carbamate pre-treatment and soman challenge with human erythrocyte and muscle homogenate AChE. Both enzyme sources were immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. AChE activity was continuously analyzed in a flow-through detector. Pre-inhibition of AChE with pyridostigmine or physostigmine resulted in a concentration-dependent increase in carbamylation, residual activity after soman inhibition and fraction of decarb-amylation AChE after discontinuation of the inhibitors without differences between human erythrocyte and muscle AChE. This data support the view that human erythrocyte AChE is an adequate surrogate marker for synaptic AChE in OP poisoning.

Electrochemical Reduction of Ellman's Reagent: A Novel Selective Detection Protocol for Thiol Compounds

AbstractHerein we detail a novel approach to the detection of total sulfhydryl thiol species based on an electrochemical adaptation of the classical Ellman's reaction. The ability to electrochemically reduce the nitro moieties of both the parent 5,5‐dithiobis(2‐nitrobenzoic acid) (DTNB) and the resulting 5‐thio‐2‐nitrobenzoic acid (TNBA) species provides a selective means of detecting thiol compounds. The use of the reductive sweep means that electrode passivation as a result of oxidative electrochemical procedures can be avoided and the selectivity improved through positioning the analytical signal within a region where few interferences reside. The electrochemical protocol enables the detection of the thiols in both acidic and basic media, which the colorimetric method is unable to do.

Fluorometric Assay for the Determination of Glutathione Reductase Activity

The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex ®-GS, 0.22 μm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax ® and Potter–Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5 h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 μM) or HI 6 (30 μM) of sarin-inhibited human muscle acetylcholinesterase (0.142 ± 0.004 min −1 and 0.166 ± 0.008 min −1, respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.

Dimerization and Actin-bundling Properties of Villin and Its Role in the Assembly of Epithelial Cell Brush Borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

Biochemical Characterization of Thepolysialic Acid-specific O-Acetyltransferase NeuO of Escherichia coli K1

Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of alpha2,8-linked polysialic acid, represents the major virulence factor. In some K1 strains, phase-variable O-acetylation of the capsular polysaccharide is observed, a modification that is catalyzed by the prophage-encoded O-acetyltransferase NeuO. Phase variation is mediated by changes in the number of heptanucleotide repeats within the 5'-coding region of neuO, and full-length translation is restricted to repeat numbers that are a multiple of three. To understand the biochemical basis of K1 capsule O-acetylation, NeuO encoded by alleles containing 0, 12, 24, and 36 repeats was expressed and purified to homogeneity via a C-terminal hexahistidine tag. All NeuO variants assembled into hexamers and were enzymatically active with a high substrate specificity toward polysialic acid with >14 residues. Remarkably, the catalytic efficiency (k(cat)/K(m)(donor)) increased linearly with increasing numbers of repeats, revealing a new mechanism for modulating NeuO activity. Using homology modeling, we predicted a three-dimensional structure primarily composed of a left-handed parallel beta-helix with one protruding loop. Two amino acids critical for catalytic activity were identified and corresponding alanine substitutions, H119A and W143A, resulted in a complete loss of activity without affecting the oligomerization state. Our results indicate that in NeuO typical features of an acetyltransferase of the left-handed beta-helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 microM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein-diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 microM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 microM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).