Annie Martel

New Plasmid-Mediated Nucleotidylation of Aminoglycoside Antibiotics in Staphylococcus aureus

A wild-type strain of Staphylococcus aureus, which inactivates a wide variety of aminoglycosides (except the gentamicin components), has been found to harbor a plasmid (RAp01) that mediates the biosynthesis of a nucleotidyltransferase. This enzyme modifies the 4′-hydroxy function of these antibiotics. The plasmid has been studied, the enzyme responsible for this resistance pattern has been isolated by affinity chromatography, and its kinetics and physicochemistry have been characterized. The target of this enzyme has also been located by demonstrating the structure of one inactivated compound, 4′-(O)-adenylyltobramycin.

(η6-Arene) ruthenium(II) complexes and metallo-papain hybrid as Lewis acid catalysts of Diels–Alder reaction in water

Covalent embedding of a (eta(6)-arene) ruthenium(II) complex into the protein papain gives rise to a metalloenzyme displaying a catalytic efficiency for a Lewis acid-mediated catalysed Diels-Alder reaction enhanced by two orders of magnitude in water.

Pyridoxal 5′phosphate binding site of Escherichia Coli beta cystathionase and cystathionine gamma synthase comparison of their sequences

The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 21 3790-3794.) showed a high degree of homology between the three PLP binding sites with the presence of the tripeptide sequence: Thr-Lys(Pxy)-Tyr in their structure. This homology suggests that these enzymes of methionine metabolism have probably the same origin.

Functionalization of nitrocellulose membranes using ammonia plasma for the covalent attachment of antibodies for use in membrane-based immunoassays

A plasma process using NH 3 was developed in order to incorporate amine functions on the surface of nitrocellulose membranes for an oriented covalent attachment of antibodies through their oxidized carbohydrate moieties. An average covalent binding capacity of 60 μg/cm 2 of antibodies was reached. The potential use of this process in developing environmental immunochemical techniques was demonstrated.

2″-O-Phosphorylation of Gentamicin Components by a Staphylococcus aureus Strain Carrying a Plasmid

A wild-type strain of Staphylococcus aureus that inactivates the 4,6-glycosidically linked deoxystreptamine aminoglycoside antibiotics by a plasmid-mediated process was found to harbor two enzymes: an acetyltransferase of the AAC(6′) type and a new phosphotranferase specific to the gentamicin components. The target of this last enzyme is the 2″-hydroxyl function of these antibiotics, since one inactivated compound is 2″-(O)-phosphorylsisomicin.

Cysteine‐Specific, Covalent Anchoring of Transition Organometallic Complexes to the Protein Papain from Carica papaya

Site‐directed and covalent introduction of various transition metal–organic entities to the active site of the cysteine endoproteinase, papain, was achieved by treatment of this enzyme with a series of organometallic maleimide derivatives specially designed for the purpose. Kinetic studies made it clear that time‐dependent irreversible inactivation of papain occurred in the presence of these organometallic maleimides as a result of Michael addition of the sulfhydryl of Cys25. The rate and mechanism of inactivation were highly dependent on the structure of the organometallic entity attached to the maleimide group. Combined ESI‐MS and IR analysis indicated that all the resulting papain adducts contained one organometallic moiety per protein molecule. This confirmed that chemospecific introduction of the metal complexes was indeed achieved. Thus, three novel reagents for heavy‐atom derivatization of protein crystals, which include ruthenium, rhenium and tungsten, are now available for the introduction of electron‐dense scatterers for phasing of X‐ray crystallographic data.

Generation and characterization of polyclonal antibodies against microcystins-Application to immunoassays and immunoaffinity sample preparation prior to analysis by liquid chromatography and UV detection.

Polyclonal antibodies against microcystin-LR (MC-LR), a cyclic heptapeptide toxin, were generated in rabbits using MC-LR-BSA. An enzyme-linked immunosorbent assay (ELISA) was developed for the characterization of the antibodies and their potential use for analytical purposes. The concentration of MC-LR that inhibits 50% of antibody-antigen binding (IC(50)) was 0.5mugL(-1) for the indirect ELISA format and 0.9mugL(-1) for the direct ELISA, using MC-LR-horseradish peroxidase conjugate. The limit of detection corresponding to IC(80) was found to be 0.06mugL(-1), well below the Word Health Organization level for drinking water of 1mugL(-1). The direct competitive ELISA was applied to water samples and was shown useful for screening purposes. The developed anti-microcystin antibodies were immobilized on solid supports for use in selective solid phase extraction (SPE) systems, prior to liquid chromatography (LC) quantification. An immunoaffinity cartridge (IAC), a Sepharose((R))-based cartridge incorporating 2mg of antibodies allowed the selective and quantitative recovery of a mixture of 0.2mug of MCs showing potential use in sample preparation of real matrices. When applied to water and green algae samples, average recoveries from Sepharose((R))-based cartridges were in the range of 86-113% for water samples and 85-92% for blue-green algae samples. Selectivity of the IAC clean-up was proven by comparison with non-specific solid phase extraction using octadecylsilica (ODS) sorbent. Results obtained using LC/UV after IAC clean-up agreed well with results obtained using liquid chromatography and mass spectrometry detection (LC/MS and LC/MS/MS) after SPE-C18 clean-up, allowing therefore to validate the resulting technique.

Development and Validation of an Indirect Enzyme Immunoassay for the Detection of the Herbicide Isoproturon in Water Matrices

Abstract An indirect enzyme immunoassay (EIA) for the detection of the phenylurea herbicide isoproturon is described. The specific antibodies did not cross-react with other structurally related compounds. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.64 ng/mL. The sensitivities were 0.07 ng/mL (IC80) and 0.02 ng/mL (IC90) respectively, when the crude serum was used in the assay. Matrix effects were observed when river water samples were analyzed showing recoveries as high as 150%. The IC50 was increased to 0.81 ng/mL. To overcome these difficulties, a novel method of anatibody purification was developed to reduce the heterogeneity of the medium when the test was performed with complex surface water matrices. This technique involved the extraction of the specific anti-isoproturon antibodies from the crude anti-serum. The refined fraction gave an IC50 not higher than 0.29 ng/mL and an IC90 of 0.01 ng/mL, when assayed with river water samples. The method was vali...

3-N Enzymatic Acetylation of Gentamicin, Tobramycin, and Kanamycin by Escherichia coli Carrying an R Factor

A new R factor-mediated gentamicin acetyltransferase obtained from a clinical isolate of gentamicin-, tobramycin-, and kanamycin-resistant Klebsiella was characterized. R factor R 176 was transferred to Escherichia coli K-12 LA 290 from a Klebsiella. Resistance transfer, minimal inhibitory concentrations of various aminoglycoside antibiotics, enzymatic inactivation, and the isoelectric point of the enzyme obtained were determined. We found that R factor R 176 could govern the enzymatic 3-N acetylation of gentamicin, tobramycin, and kanamycin, and isoelectric point determinations suggested that the enzymes governed by R 176 and R 135 are different. The genes responsible for resistance to gentamicin, tobramycin, and kanamycin govern the synthesis of an isoenzyme of AAC(3)I, which we propose to call AAC(3)II.

Mass Spectrometric Determination of the Cleavage Sites inEscherichia coli Dihydroorotase Induced by a Cysteine-specific Reagent

Escherichia coli dihydroorotase contains six cysteines/subunit, which are potential ligands of structural and catalytic zinc metals at protein sites of the enzyme. Specific thiol reagents modify, in nondenaturing conditions only, two of these cysteines; these two residues are thought to be ligands of structural zinc. We report here on the localization of these two cysteines on the polypeptide chain through their cyanylation by 2-nitro-5-thiocyanobenzoic acid (NTCB) and the analysis by mass spectrometry of the protein adducts. This is the first study ofE. coli dihydroorotase by mass spectrometry, allowing the accurate determination of the subunit molecular weight (38,695). Treatment of dihydroorotase by NTCB induced a cleavage N-terminal to the cyanylated cysteines. The resulting fragments visualized on electrophoresis gel have been N-terminal sequenced, and their masses were determined by electrospray-ionizing mass spectrometry. This allowed the identification of cysteines 221 and 265 as the two residues cyanylated by the reagent NTCB. Results from gel filtration of dihydroorotase cyanylated on the two cysteines indicate that these residues are involved in subunit interactions leading to the active dimer. Consistent with literature data, we assume that cysteine 221 and cysteine 265, along with the neighboring cysteines 263 and 268 arranged in cluster, are potential ligands of structural zinc of E. coli dihydroorotase.