80 results on '"Nicole Moguilevsky"'
Search Results
2. Exposure of endothelial cells to physiological levels of myeloperoxidase-modified LDL delays pericellular fibrinolysis.
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Karim Zouaoui Boudjeltia, Jalil Daher, Pierre Van Antwerpen, Nicole Moguilevsky, Paul Delree, Jean Ducobu, Martine Raes, Bassam Badran, Michel Vanhaeverbeek, Dany Brohee, Claude Remacle, and Luc Vanhamme more...
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Medicine ,Science - Abstract
BACKGROUND: Blood fluidity is maintained by a delicate balance between coagulation and fibrinolysis. The endothelial cell surface is a key player in this equilibrium and cell surface disruptions can upset the balance. We investigated the role of pericellular myeloperoxidase oxidized LDLs (Mox-LDLs) in this balance. METHODS AND RESULTS: We designed a technical device that enabled us to monitor fibrinolysis in real-time at the surface of an endothelial cell line (EA.hy926), and showed that Mox-LDL decreased pericellular fibrinolysis. There were no changes in fibrinolysis when EA.hy926 endothelial cells were exposed to native LDL (24 hours) at doses of 10, 50, 100 and up to 1250 µg/ml. However, treatment of EA.hy926 endothelial cells with 10 and 50 µg/ml of Mox-LDL (physiological serum concentrations) increased the lysis time by 15 and 13%, respectively (p more...
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- 2012
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Catalog
3. Temporal dissociation between myeloperoxidase (MPO)-modified LDL and MPO elevations during chronic sleep restriction and recovery in healthy young men.
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Karim Zouaoui Boudjeltia, Brice Faraut, Maria José Esposito, Patricia Stenuit, Michal Dyzma, Pierre Van Antwerpen, Dany Brohée, Luc Vanhamme, Nicole Moguilevsky, Michel Vanhaeverbeek, and Myriam Kerkhofs more...
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Medicine ,Science - Abstract
OBJECTIVES: Many studies have evaluated the ways in which sleep disturbances may influence inflammation and the possible links of this effect to cardiovascular risk. Our objective was to investigate the effects of chronic sleep restriction and recovery on several blood cardiovascular biomarkers. METHODS AND RESULTS: Nine healthy male non-smokers, aged 22-29 years, were admitted to the Sleep Laboratory for 11 days and nights under continuous electroencephalogram polysomnography. The study consisted of three baseline nights of 8 hours sleep (from 11 pm to 7 am), five sleep-restricted nights, during which sleep was allowed only between 1 am and 6 am, and three recovery nights of 8 hours sleep (11 pm to 7 am). Myeloperoxidase-modified low-density lipoprotein levels increased during the sleep-restricted period indicating an oxidative stress. A significant increase in the quantity of slow-wave sleep was measured during the first recovery night. After this first recovery night, insulin-like growth factor-1 levels increased and myeloperoxidase concentration peaked. CONCLUSIONS: We observed for the first time that sleep restriction and the recovery process are associated with differential changes in blood biomarkers of cardiovascular disease. more...
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- 2011
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4. Human peroxidasin 1 promotes angiogenesis through ERK1/2, Akt, and FAK pathways
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Michael Herfs, Mohamed Amri, Pierre Van Antwerpen, Hayfa Medfai, Paul G. Furtmüller, Christian Obinger, Benjamin Sevcnikar, Vincent Castronovo, Olivier Peulen, Vincent Nuyens, Luc Vanhamme, Alia Khalil, Karim Zouaoui Boudjeltia, Nicole Moguilevsky, Alexandre Rousseau, and Martina Paumann-Page more...
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0301 basic medicine ,Physiology ,Angiogenesis ,Neovascularization, Physiologic ,Chick Embryo ,030204 cardiovascular system & hematology ,Cell Line ,Focal adhesion ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Physiology (medical) ,Animals ,Humans ,Phosphorylation ,Protein kinase B ,Cell Proliferation ,Mitogen-Activated Protein Kinase 1 ,Tube formation ,Mitogen-Activated Protein Kinase 3 ,PDGFB ,Chemistry ,Endothelial Cells ,Sciences bio-médicales et agricoles ,Cell biology ,Enzyme Activation ,030104 developmental biology ,Gene Expression Regulation ,Peroxidases ,Focal Adhesion Kinase 1 ,Signal transduction ,Cardiology and Cardiovascular Medicine ,Proto-Oncogene Proteins c-akt ,Signal Transduction ,Platelet-Derived Growth Factor Subunit B - Abstract
The term angiogenesis refers to sprouting of new blood vessels from pre-existing ones. The angiogenic process involves cell migration and tubulogenesis requiring interaction between endothelial cells and the extracellular matrix. Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase found embedded in the basement membranes. As it promotes the stabilization of extracellular matrix, we investigated its possible role in angiogenesis both in vitro and in vivo., SCOPUS: ar.j, info:eu-repo/semantics/published more...
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- 2018
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5. Myeloperoxidase-catalyzed oxidation of cyanide to cyanate
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Michel Vanhaeverbeek, Monika Soudi, Vincent Nuyens, Jean Ducobu, Catherine Coremans, Cédric Delporte, Nicole Moguilevsky, Karim Zouaoui Boudjeltia, Damien Dufour, Marc Dieu, Pierre Van Antwerpen, Wanda F. Reynolds, Bernard Robaye, Florence Reye, Alexandre Rousseau, Paul G. Furtmüller, Christian Obinger, Martine Raes, Caroline Noyon, Luc Vanhamme, Richard A. Maki, and Jean Neve more...
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0301 basic medicine ,Hemeprotein ,Hypochlorous acid ,post-translational modification (PTM) ,Cyanide ,Lysine ,030204 cardiovascular system & hematology ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,cardiovascular disease ,Animals ,Humans ,Molecular Biology ,Cyanates ,Peroxidase ,Homocitrulline ,Mice, Knockout ,Cyanides ,Protein Carbamylation ,Thiocyanate ,biology ,Chemistry ,lipoprotein ,Cell Biology ,Sciences bio-médicales et agricoles ,Cyanate ,Plaque, Atherosclerotic ,myeloperoxidase ,030104 developmental biology ,Myeloperoxidase ,biology.protein ,Enzymology ,Citrulline ,atherosclerosis ,Oxidation-Reduction - Abstract
Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase, which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine and low-density-lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high fat diet and carrying human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homo-citrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques., SCOPUS: ar.j, info:eu-repo/semantics/published more...
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- 2018
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6. Simultaneous measurement of protein-bound 3-chlorotyrosine and homocitrulline by LC-MS/MS after hydrolysis assisted by microwave
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Didier Serteyn, Luc Vanhamme, Thierry Franck, Philippe Madhoun, Nicole Moguilevsky, Caroline Noyon, Karim Zouaoui Boudjeltia, Jean Neve, Alexandre Rousseau, Joëlle Nortier, Cédric Delporte, Michel Vanhaeverbeek, Pierre Van Antwerpen, Martine Raes, Jean-Marc Desmet, and Damien Dufour more...
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Male ,Time Factors ,medicine.medical_treatment ,Context (language use) ,Pharmacology ,medicine.disease_cause ,Analytical Chemistry ,chemistry.chemical_compound ,Renal Dialysis ,Tandem Mass Spectrometry ,medicine ,Animals ,Humans ,Microwaves ,Aged ,Peroxidase ,3-Chlorotyrosine ,Aged, 80 and over ,Homocitrulline ,biology ,Chemistry ,Hydrolysis ,Lysine ,Blood Proteins ,Middle Aged ,medicine.disease ,Uremia ,Transplantation ,Biochemistry ,Myeloperoxidase ,biology.protein ,Citrulline ,Tyrosine ,Female ,Hemodialysis ,Blood Chemical Analysis ,Oxidative stress ,Chromatography, Liquid - Abstract
A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation is not possible, uremia can be reduced by hemodialysis although the repeated interventions have been implicated in loss of renal function, partially as a result of chronic inflammation and/or oxidative stress processes. In this context, it has been suggested that myeloperoxidase (MPO) can contribute to the oxidative stress during hemodialysis and to the cardiovascular risk. Protein damages due to MPO activity have never been assessed during hemodialysis although two of its reaction products, 3-chlorotyrosine and homocitrulline, are of interest. Indeed, the first one is a specific product of MPO activity and the formation of the second one could be catalyzed by MPO. In order to analyze these products in plasma proteins, a total hydrolysis method followed by liquid chromatography mass spectrometry analysis was developed. Different conditions of hydrolysis were tested and the optimized procedure was assessed for complete hydrolysis and artifactual chlorination. Finally, the method was used for analyzing 3-chlorotyrosine and homocitrulline in plasma proteins during a hemodialysis session in fifteen patients and data were related to measurements of MPO concentration and activity. Both increases in MPO activity and protein-bound 3-chlorotyrosine were observed, highlighting the involvement of MPO in oxidative stress during hemodialysis and further demonstrating the link between hemodialysis and cardiovascular diseases. more...
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- 2012
7. Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase
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Martine Raes, Jean Nève, Jean-Claude Michalski, Nicole Moguilevsky, Pierre Van Antwerpen, Cédric Delporte, Damien Calay, Valegh Faid, Marie-Christine Slomianny, Alexandre Rousseau, Luc Vanhamme, Karim Zouaoui Boudjeltia, Michel Vanhaeverbeek, Christian Obinger, and Paul G. Furtmüller more...
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chemistry.chemical_classification ,Glycosylation ,biology ,Chinese hamster ovary cell ,Cell Biology ,biology.organism_classification ,Biochemistry ,Molecular biology ,law.invention ,carbohydrates (lipids) ,chemistry.chemical_compound ,Enzyme ,Biosynthesis ,chemistry ,law ,Myeloperoxidase ,biology.protein ,Recombinant DNA ,Cricetulus ,Molecular Biology ,Peroxidase - Abstract
The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the three-dimensional structure of MPO. more...
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- 2010
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8. Resveratrol Inhibits the Activity of Equine Neutrophil Myeloperoxidase by a Direct Interaction with the Enzyme
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Maurice Lamy, Pierre Van Antwerpen, Ginette Deby-Dupont, Ange Mouithys-Mickalad, Stephan Kohnen, Nicole Moguilevsky, Didier Serteyn, Thierry Franck, Karim Zouaoui Boudjeltia, and Christiane Deby
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Antioxidant ,endocrine system diseases ,Neutrophils ,Neutrophile ,medicine.medical_treatment ,Resveratrol ,chemistry.chemical_compound ,Stilbenes ,medicine ,Animals ,Horses ,Enzyme Inhibitors ,Peroxidase ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Chemistry ,organic chemicals ,Degranulation ,food and beverages ,General Chemistry ,Enzyme ,Biochemistry ,Myeloperoxidase ,biology.protein ,Reactive Oxygen Species ,General Agricultural and Biological Sciences - Abstract
Resveratrol is a polyphenolic antioxidant present in beverage and food known for its multiple protective effects. We report the inhibitory effects of resveratrol on equine myeloperoxidase (MPO), a hemic peroxidase present in the granules of the neutrophils involved in the inflammatory response. Resveratrol inhibited the production of reactive oxygen species (ROS) by stimulated equine neutrophils by acting as a direct scavenger of the ROS released by the cells but did not modify the degranulation of the stimulated neutrophils as the amounts of released MPO were unchanged. Resveratrol strongly inhibited the chlorination, oxidation, and nitration activities of MPO in a dose-dependent manner. By an original technique of specific immunological extraction followed by enzymatic detection (SIEFED), we demonstrated that resveratrol inhibited the peroxidasic activity of the MPO measured by a direct interaction such as the fixation of resveratrol on the enzyme. The observation of a decrease of the accumulation of compound II suggested that resveratrol acts as an electron donor for MPO reduction. more...
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- 2007
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9. Disruption of the Aspartate to Heme Ester Linkage in Human Myeloperoxidase
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Martina Zederbauer, Christa Jakopitsch, Johanna Stampler, Nicole Moguilevsky, Paul G. Furtmüller, Marzia Bellei, Christian Obinger, and Gianantonio Battistuzzi
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biology ,Thiocyanate ,Chemistry ,Stereochemistry ,Active site ,Cell Biology ,Biochemistry ,Cofactor ,chemistry.chemical_compound ,Bromide ,Covalent bond ,Myeloperoxidase ,biology.protein ,Molecular Biology ,Heme ,Pyrrole - Abstract
In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp94 to heme ester bond decreased the one-electron reduction potential E′0 [Fe(III)/Fe(II)] from 1 to –55 mV at pH 7.0 and 25 °C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp94 to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands. more...
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- 2007
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10. Expression, purification, and structural prediction of the Ets transcription factor ERM
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Vincent Raussens, Yvan de Launoit, Nicole Moguilevsky, Jean Marie Ruysschaert, Carine Van Lint, Catherine Tricot, Isabelle Huvent, Jean-Luc Baert, Sébastien Mauen, and Dominique Demonte
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Spectrophotometry, Infrared ,Protein Conformation ,Blotting, Western ,Molecular Sequence Data ,Biophysics ,Sf9 ,Spodoptera ,Biochemistry ,DNA-binding protein ,Protein structure ,Affinity chromatography ,Amino Acid Sequence ,Molecular Biology ,Transcription factor ,Peptide sequence ,DNA Primers ,Base Sequence ,biology ,Circular Dichroism ,ETS transcription factor family ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,DNA-Binding Proteins ,Electrophoresis, Polyacrylamide Gel ,Transcription Factors - Abstract
The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors. Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an alpha-helix. In contrast, the 250-residue central domain seems to have very little structure. more...
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- 2006
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11. Role of the Covalent Glutamic Acid 242−Heme Linkage in the Formation and Reactivity of Redox Intermediates of Human Myeloperoxidase
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Paul G. Furtmüller, Martina Zederbauer, Karin Neugschwandtner, Christian Obinger, Christa Jakopitsch, Walter Jantschko, and Nicole Moguilevsky
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Bromides ,Eosinophil Peroxidase ,Hypochlorous acid ,Stereochemistry ,Sulfonium ,Glutamine ,Glutamic Acid ,CHO Cells ,Heme ,Ferric Compounds ,Biochemistry ,chemistry.chemical_compound ,Methionine ,Chlorides ,Cricetinae ,Enzyme Stability ,Animals ,Humans ,Organic chemistry ,Peroxidase ,Pyrrole ,Aspartic Acid ,Binding Sites ,Cyanides ,biology ,Circular Dichroism ,Halogenation ,Recombinant Proteins ,chemistry ,Covalent bond ,Myeloperoxidase ,biology.protein ,Oxidation-Reduction - Abstract
In human myeloperoxidase the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243 and the beta-carbon of the vinyl group on pyrrole ring A. In the present study, wild-type recombinant myeloperoxidase (recMPO) and the variant Glu242Gln were produced in Chinese hamster ovary cells and investigated in a comparative sequential-mixing stopped-flow study in order to elucidate the role of the Glu242-heme ester linkage in the individual reaction steps of both the halogenation and peroxidase cycle. Disruption of the ester bond increased heme flexibility, blue shifted the UV-vis spectrum, and, compared with recMPO, decelerated cyanide binding (1.25 x 10(4) versus 1.6 x 10(6) M(-)(1) s(-)(1) at pH 7 and 25 degrees C) as well as compound I formation mediated by either hydrogen peroxide (7.8 x 10(5) versus 1.9 x 10(7) M(-)(1) s(-)(1)) or hypochlorous acid (7.5 x 10(5) versus 2.3 x 10(7) M(-)(1) s(-)(1)). The overall chlorination and bromination activity of Glu242Gln was 2.0% and 24% of recMPO. The apparent bimolecular rate constants of compound I reduction by chloride (65 M(-)(1) s(-)(1)), bromide (5.4 x 10(4) M(-)(1) s(-)(1)), iodide (6.4 x 10(5) M(-)(1) s(-)(1)), and thiocyanate (2.2 x10(5) M(-)(1) s(-)(1)) were 500, 25, 21, and 63 times decreased compared with recMPO. By contrast, Glu242Gln compound I reduction by tyrosine was only 5.4 times decreased, whereas tyrosine-mediated compound II reduction was 60 times slower compared with recMPO. The effects of exchange of Glu242 on electron transfer reactions are discussed. more...
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- 2005
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12. Alveolar Macrophage Activation by Myeloperoxidase
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John A. Lincoln, Stanley S. Lefkowitz, Herman Friedman, Alex Bollen, Doris L. Lefkowitz, Erin Roberts, Ken Grattendick, Nicole Moguilevsky, and Rodney Stuart
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Pulmonary and Respiratory Medicine ,Clinical Biochemistry ,Enzyme-Linked Immunosorbent Assay ,Inflammation ,Proinflammatory cytokine ,Mice ,Phagocytosis ,Downregulation and upregulation ,Macrophages, Alveolar ,medicine ,Animals ,Macrophage ,Molecular Biology ,Peroxidase ,biology ,Chemistry ,Pneumonia ,Cell Biology ,Macrophage Activation ,Rats ,Respiratory burst ,Rats, Inbred Lew ,Myeloperoxidase ,Immunology ,Alveolar macrophage ,biology.protein ,Cytokines ,Female ,Tumor necrosis factor alpha ,medicine.symptom - Abstract
Inflammation of the lung is characterized by the influx of increased numbers of various leukocytes including polymorphonuclear leukocyte (PMN) neutrophils. In addition to cells, numerous studies have pointed to the role of tumor necrosis factor-alpha in the inflammatory process. This study addresses a previously unrecognized interaction between neutrophil-derived myeloperoxidase (MPO) and resident alveolar macrophages (AMø). Rat AMø exposed to either enzymatically active recombinant MPO or enzymatically inactive MPO (iMPO) exhibited an increased respiratory burst (RB). When iMPO was employed, the enhancement of the RB was greater than that observed with MPO. Although the RB was greater with iMPO, macrophage (Mø)-mediated intracellular candidic activity was equivalent for both MPO and iMPO. It is known that pro- inflammatory cytokines contribute to the inflammatory process. When rat AMø were exposed to both forms of myeloperoxidase, iMPO demonstrated greater upregulation of cytokine genes as well as product. These data suggest that at the site of inflammation, neutrophil-derived MPO and iMPO stimulate AMø, resulting in an increased inflammatory and cytotoxic state, and thereby contributing to the general lung inflammatory response. more...
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- 2002
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13. MYELOPEROXIDASE AND ITS PRODUCTS IN SYNOVIAL FLUID OF PATIENTS WITH TREATED OR UNTREATED RHEUMATOID ARTHRITIS
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Jean Neve, Alexandre Rousseau, Van Antwerpen, Didier Serteyn, Caroline Noyon, Thierry Franck, Luc Vanhamme, Martine Raes, Nicole Moguilevsky, Cédric Delporte, Patrick Durez, Michel Vanhaeverbeek, A. Nzeusseu Toukap, and K. Zouaoui Boudjeltia more...
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Male ,medicine.medical_specialty ,Chimie analytique ,Arthritis ,Inflammation ,Osteoarthritis ,Sciences biomédicales en général ,Biochemistry ,Arthritis, Rheumatoid ,chemistry.chemical_compound ,Internal medicine ,Synovial Fluid ,medicine ,Humans ,Synovial fluid ,Chloro-tyrosine ,Peroxidase ,Homocitrulline ,Interleukine-8 ,Myeloperoxidase ,biology ,business.industry ,Interleukin-8 ,Interleukin ,General Medicine ,medicine.disease ,Endocrinology ,chemistry ,Rheumatoid arthritis ,Immunology ,Orthopédie ,biology.protein ,Cytokines ,Female ,medicine.symptom ,business - Abstract
Objective Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), IL-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated. Methods Patients (n=105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. DAS-28 was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine, homocitrulline, IL-8 and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum. Results DAS-28 and CRP level were not significantly different between groups. MPO activity, and MPO, chloro-tyrosine and homocitrulline levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. Conclusions MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and homocitrulline in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published more...
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- 2014
14. Human recombinant myeloperoxidase antiviral activity on cytomegalovirus
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S. Fourez, N. Tasiaux, Nicole Moguilevsky, Andrew W. Bollen, Lise Thiry, A.-M. Verheyden, and K. El Messaoudi
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Infectivity ,Human cytomegalovirus ,biology ,Cell growth ,Congenital cytomegalovirus infection ,medicine.disease ,Virology ,law.invention ,Infectious Diseases ,Antigen ,law ,Myeloperoxidase ,biology.protein ,medicine ,Recombinant DNA ,Intracellular - Abstract
In vitro incubation of human cytomegalovirus (Towne strain) with 8 U/ml human recombinant myeloperoxidase plus sodium chloride and glucose nearly abolished viral infectivity. To assay the effect on intracellular infection, cell toxicity of the enzymes was first studied. Even the high dose of 16 U/ml of recombinant myeloperoxidase plus 10 mU/ml glucose oxidase did not decrease MRC5 cell growth. By contrast, this dose reduced proliferation of activated THP1 cells. Even half of the myeloperoxidase dose proved slightly toxic to these cells. Non-cytotoxic concentrations of the reagents were used to monitor their effect on cytomegalovirus infection. In MRC5 cells, even the low dose of 4 U/ml myeloperoxidase plus glucose oxidase inhibited synthesis of cytomegalovirus early antigens, as tested by immunofluorescence. Viral release in the supernatant was decreased by 4 logs. In THP1 cells, which produce endogenously hydrogen peroxide, myeloperoxidase alone (8 U/ml) decreased the formation of early and late antigens by 53 and 44%, respectively. more...
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- 2001
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15. A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase
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Günther Regelsberger, Nicole Moguilevsky, Christa Jakopitsch, Paul G. Furtmüller, Walter Jantschko, and Christian Obinger
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Iodide ,Biophysics ,CHO Cells ,In Vitro Techniques ,Stopped-flow spectroscopy ,Biochemistry ,Recombinant myeloperoxidase ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Bromide ,Catalytic Domain ,Cricetinae ,Genetics ,medicine ,Animals ,Humans ,Ascorbate ,Protein Structure, Quaternary ,Hydrogen peroxide ,Halide ,Molecular Biology ,Peroxidase ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chinese hamster ovary cell ,030302 biochemistry & molecular biology ,Active site ,Compound II ,Hydrogen Peroxide ,Cell Biology ,Recombinant Proteins ,Hypochlorous Acid ,Kinetics ,Enzyme ,chemistry ,Compound I ,Myeloperoxidase ,biology.protein ,Ferric ,Protein Processing, Post-Translational ,medicine.drug - Abstract
Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi-mixing stopped-flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9 x 10(7) M(-1) s(-1), pH 7 and 15 degrees C) and reduction of compound I to compound II (3.0 x 10(4) M(-1) s(-1), pH 7 and 15 degrees C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6 x 10(4) M(-1) s(-1), 1.4 x 10(6) M(-1) s(-1), 1.4 x 10(7) M(-1) s(-1) and 1.4 x 10(7) M(-1) s(-1), respectively), whereas the endogenous one-electron donor ascorbate mediated transformation of compound I to compound II (2.3 x 10(5) M(-1) s(-1)) and of compound II back to the resting enzyme (5.0 x 10(3) M(-1) s(-1)). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently. more...
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- 2001
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16. Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs investigated with a human recombinant enzyme
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Nathalie Parij, Jean Neve, and Nicole Moguilevsky
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Drug ,Taurine ,Antioxidant ,medicine.medical_treatment ,media_common.quotation_subject ,chemistry.chemical_compound ,polycyclic compounds ,medicine ,Humans ,IC50 ,Peroxidase ,media_common ,Pharmacology ,chemistry.chemical_classification ,Reactive oxygen species ,Dose-Response Relationship, Drug ,biology ,Anti-Inflammatory Agents, Non-Steroidal ,Recombinant Proteins ,Hypochlorous Acid ,Enzyme ,Mechanism of action ,chemistry ,Biochemistry ,Myeloperoxidase ,biology.protein ,Chlorine ,medicine.symptom - Abstract
Non-steroidal anti-inflammatory drugs (NSAIDs) were investigated for their ability to affect the chlorinating activity of human myeloperoxidase and to scavenge HOCl, the main myeloperoxidase system product. Fourteen drugs representative of various NSAIDs families were tested with the chlorination of taurine used as a detection system. All were unable to inhibit taurine chlorination in a system without myeloperoxidase. In contrast, most of them induced a dose-dependent inhibition of the taurine chlorination mediated by a myeloperoxidase/H2O2/Cl- system. This took place at variable drug concentrations and IC50 were calculated. The inhibitory effect was therefore due to a direct interaction with the enzyme rather than to HOCl scavenging. A spectroscopic method used to measure the myeloperoxidase compound II lifetime in presence of the different drugs showed that all the drugs, which inhibited chlorination activity were able to induce accumulation of compound II. The extent of chlorinating activity inhibition (IC50) was inversely related to the duration of the block of enzyme in compound II form. This further demonstrates that myeloperoxidase is an interesting target for anti-inflammatory therapy. The recombinant myeloperoxidase used for the first time in this kind of study was as convenient for pharmacological purposes as the purified one. more...
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- 2001
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17. Bovine Lactoperoxidase and Its Recombinant: Comparison of Structure and Some Biochemical Properties
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Haruto Kumura, Shingo Nakamura, Alex Bollen, Shikiko Watanabe, Nicole Moguilevsky, Kei-ichi Shimazaki, and Shinya Murata
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Circular dichroism ,Protein Conformation ,Biophysics ,Peptide ,Biochemistry ,Hydrolysate ,law.invention ,Structure-Activity Relationship ,law ,Cricetinae ,Animals ,Lactoperoxidase ,Molecular Biology ,Peptide sequence ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Chromatography ,Chinese hamster ovary cell ,Cell Biology ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Amino acid ,chemistry ,Recombinant DNA ,Cattle - Abstract
Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined. more...
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- 2000
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18. The Endothelium and Cytokine Secretion: The Role of Peroxidases as Immunoregulators
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Christopher J. Schwab, Erin Roberts, Alex Bollen, Nicole Moguilevsky, R. W. Stuart, John A. Lincoln, Stanley S. Lefkowitz, Robert C. Allen, Doris L. Lefkowitz, and Ken Grattendick
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Umbilical Veins ,Eosinophil Peroxidase ,Swine ,medicine.medical_treatment ,Immunology ,Proinflammatory cytokine ,medicine ,Animals ,Humans ,RNA, Messenger ,Interleukin 6 ,Cells, Cultured ,Peroxidase ,Respiratory Burst ,biology ,Interleukin-6 ,Interleukin-8 ,Granulocyte-Macrophage Colony-Stimulating Factor ,Molecular biology ,Up-Regulation ,Respiratory burst ,Cytokine ,Granulocyte macrophage colony-stimulating factor ,Peroxidases ,Myeloperoxidase ,biology.protein ,Cytokines ,Cytokine secretion ,Endothelium, Vascular ,Reactive Oxygen Species ,Eosinophil peroxidase ,medicine.drug - Abstract
The endothelium is frequently exposed to many proinflammatory mediators. The present study was done to determine the effects of human recombinant myeloperoxidase (MPO) and porcine eosinophil peroxidase (EPO) on certain endothelial cell (HUVEC) functions. The following areas were evaluated: (1) production of reactive oxygen intermediates (ROI), (2) cytokine secretion, and (3) regulation of mRNA cytokine transcripts. Both MPO and EPO induced the production of ROI, but an enzymatically inactive form of MPO (iMPO) was the most effective. Enzymatically inactive MPO, but not MPO, induced the secretion of interleukins 6 and 8 and granulocyte-monocyte colony-stimulating factor. A ribonuclease protection assay indicated that both iMPO and MPO upregulated mRNA cytokine transcripts; however, the former was markedly more effective. The simultaneous addition of EPO and iMPO resulted in a decrease in cytokine-specific mRNA. These data indicate a major role for peroxidases in the regulation of inflammation. more...
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- 2000
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19. A Human Milk Factor Susceptible to Cathepsin D Inhibitors Enhances Human Immunodeficiency Virus Type 1 Infectivity and Allows Virus Entry into a Mammary Epithelial Cell Line
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Nicole Van Tieghem, Nicole Moguilevsky, Corinne Liesnard, Lise Thiry, Kamal El Messaoudi, and Alex Bollen
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viruses ,Lymphocyte ,Immunology ,Gene Expression ,Cathepsin D ,Galactosylceramides ,HIV Envelope Protein gp120 ,Biology ,Microbiology ,CXCR4 ,Virus ,chemistry.chemical_compound ,Receptors, HIV ,Viral entry ,Virology ,Pepstatins ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Breast ,RNA, Messenger ,skin and connective tissue diseases ,Cathepsin ,Infectivity ,virus diseases ,Epithelial Cells ,Molecular biology ,Virus-Cell Interactions ,Milk ,medicine.anatomical_structure ,chemistry ,Insect Science ,HIV-1 ,RNA, Viral ,Female ,Pepstatin - Abstract
Human immunodeficiency virus type 1 (HIV-1) growth in lymphocyte cultures was increased when the virus inoculum was incubated in breast milk. The enhancing effect of milk was abolished by anti-cathepsin D antibody or by pepstatin A, a cathepsin D inhibitor. The cathepsin D-producing CD4-negative MCF7 mammary cells supported the growth of some HIV-1 isolates. An MCF7 line chronically producing HIV-1 IIIb was obtained. Cathepsin D may induce conformational modification of viral gp120, allowing direct interaction with a coreceptor. We demonstrated the presence of CXCR4 mRNA in MCF7 cells. more...
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- 2000
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20. The Met243 sulfonium ion linkage is responsible for the anomalous magnetic circular dichroism and optical spectral properties of myeloperoxidase
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Michael K. Johnson, Ingeborg M. Kooter, Alex Bollen, Nicole Moguilevsky, Brian P. Koehler, Ron Wever, and SILS Other Research (FNWI)
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Hemeprotein ,Protein Conformation ,Sulfonium ,Sulfonium Compounds ,Photochemistry ,Ferric Compounds ,Biochemistry ,law.invention ,Inorganic Chemistry ,Fluorides ,Magnetics ,chemistry.chemical_compound ,law ,Humans ,Ferrous Compounds ,Electron paramagnetic resonance ,Heme ,Peroxidase ,Cyanides ,biology ,Chemistry ,Magnetic circular dichroism ,Circular Dichroism ,Lactoperoxidase ,Covalent bond ,Myeloperoxidase ,biology.protein ,Spectrophotometry, Ultraviolet - Abstract
The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6-273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243. more...
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- 1999
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21. Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality☆
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Patrick Robberecht, E. Di Paolo, Alex Bollen, Jean-Pierre Vilardaga, H. Petry, Nicole Moguilevsky, and Magali Waelbroeck
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Physiology ,Molecular Sequence Data ,Vasoactive intestinal peptide ,Secretin receptor family ,CHO Cells ,Secretin family ,Biology ,digestive system ,Biochemistry ,Protein Structure, Secondary ,Receptors, G-Protein-Coupled ,Receptors, Gastrointestinal Hormone ,Secretin ,Cellular and Molecular Neuroscience ,Endocrinology ,Cricetinae ,Animals ,Amino Acid Sequence ,Amino Acids ,Receptor ,Conserved Sequence ,chemistry.chemical_classification ,Ligand (biochemistry) ,Rats ,Amino acid ,chemistry ,Mutagenesis, Site-Directed ,Receptors, Vasoactive Intestinal Peptide ,Secretin receptor ,hormones, hormone substitutes, and hormone antagonists ,Adenylyl Cyclases - Abstract
The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties. more...
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- 1999
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22. Properties of a Recombinant Human Secretin Receptor
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Alex Bollen, P. De Neef, Magali Waelbroeck, H. Petry, E. Di Paolo, Nicole Moguilevsky, Patrick Robberecht, and Johnny Cnudde
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DNA, Complementary ,Endocrinology, Diabetes and Metabolism ,Molecular Sequence Data ,Vasoactive intestinal peptide ,Secretin receptor family ,CHO Cells ,Secretin family ,Biology ,Transfection ,digestive system ,Receptors, G-Protein-Coupled ,Receptors, Gastrointestinal Hormone ,Secretin ,Endocrinology ,Species Specificity ,Cricetinae ,Internal Medicine ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Receptor ,Pancreas ,Gene Library ,Hepatology ,Chinese hamster ovary cell ,Recombinant Proteins ,Rats ,Gastrointestinal hormone ,Biochemistry ,Secretin receptor ,Rabbits ,hormones, hormone substitutes, and hormone antagonists ,Adenylyl Cyclases ,Vasoactive Intestinal Peptide - Abstract
A secretin receptor was cloned from a commercial human pancreatic complementary DNA (cDNA) bank. The amino acid sequence deduced from the nucleotide sequence differed slightly from the three different sequences previously published, suggesting a genetic polymorphism of the human receptor. The binding properties of the receptor were evaluated by testing natural secretin, related peptides, and synthetic analogs or fragments on membranes of Chinese hamster ovary (CHO) cells expressing the receptor after transfection. The second-messenger coupling was evaluated by adenylate cyclase measurement. The human secretin receptor was compared with the rat and the rabbit receptors. In the three animals species, rat and human secretin were equipotent; rabbit secretin was equipotent on human and rabbit secretin receptors and less potent on the rat receptor. Similar data were obtained for the [Arg 16 ]-secretin analog. Deletion of histidine 1 and replacement of aspartate 3 reduced the affinity of the peptides for the three receptors; however, the reduction was more pronounced on rat than on human and rabbit secretin receptors. Finally, the low affinity of the rat and human receptors for vasoactive intestinal peptide (VIP) was identical; the rabbit receptor, however, had a 20-fold higher affinity. Thus the human secretin receptor shows properties of both rat and rabbit receptors. Evaluation of the properties of chimeric receptors will be useful to fit the ligand on the receptors. more...
- Published
- 1999
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23. Recombinant bovine lactoperoxidase as a tool to study the heme environment in mammalian peroxidases
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Francesca Varsalona, Shikiko Watanabe, Nicole Moguilevsky, Yung-Choon Yoo, Kei-ichi Shimazaki, Alex Bollen, and J.-P. Guillaume
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DNA, Complementary ,Glycosylation ,Peroxidase activity ,Molecular Sequence Data ,Biophysics ,CHO Cells ,Heme ,Biochemistry ,law.invention ,chemistry.chemical_compound ,Structural Biology ,law ,Cricetinae ,Genetics ,Animals ,Chlorination ,Amino Acid Sequence ,Lactoperoxidase ,Molecular Biology ,Recombinant lactoperoxidase ,Soret peak ,Site-directed mutagenesis ,Myeloperoxidase ,biology ,Chinese hamster ovary cell ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Peroxidases ,chemistry ,COS Cells ,biology.protein ,Recombinant DNA ,Cattle ,Peroxidase - Abstract
The cDNA encoding bovine lactoperoxidase (LPO) has been expressed in CHO cells. The recombinant LPO was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. rLPO exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Engineering of rLPO into a myeloperoxidase (MPO)-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in MPO. However, the resulting bovine LPO mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of MPO, underlining the complex nature of interactions in the heme vicinity. more...
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- 1998
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24. Enhancement of macrophage-mediated bactericidal activity by macrophage-mannose receptor-ligand interaction
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Dorls L Lefkowitz, John A. Lincoln, Nicole Moguilevsky, Alex Bollen, and Stanley S. Lefkowitz
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Male ,Neutrophils ,Phagocytosis ,Immunology ,Serum albumin ,Receptors, Cell Surface ,Mice ,Escherichia coli ,Animals ,Immunology and Allergy ,Macrophage ,Lectins, C-Type ,Receptor ,Peroxidase ,Mannan-binding lectin ,biology ,Chemistry ,Macrophages ,Serum Albumin, Bovine ,Cell Biology ,Ligand (biochemistry) ,Molecular biology ,Mice, Inbred C57BL ,Mannose-Binding Lectins ,Myeloperoxidase ,biology.protein ,Female ,Reactive Oxygen Species ,Mannose Receptor ,Mannose receptor - Abstract
Neutrophils represent one of the host's primary defenses against invading organisms. These cells often arrive at the site of infection prior to macrophages (M phi). Neutrophils release myeloperoxidase (MPO) into the micro-environment during phagocytosis. Previous studies by the present investigators have shown that M phi bactericidal activity is enhanced by exposure to MPO. A recent report suggests that as much as 40% of this protein is enzymatically inactive once it is released into the micro-environment. In the present study, exposure of M phi to an enzymatically inactive form of MPO (iMPO) or another mannosylated protein, mannosylated bovine serum albumin (mBSA), can induce the same enhanced Mø-mediated bacterial cell killing observed with the active form of MPO. Furthermore, this phenomenon is limited as galactosylated BSA (gBSA) did not induce enhancement of bacterial killing. The data suggest that interaction of either enzymatically active or inactive mannosylated proteins with the M phi mannose receptor (MMR), is sufficient to enhance M phi bactericidal activity and further underscores the binding of the MMR and resultant responses as a major host defense mechanism. more...
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- 1997
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25. Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group
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Cees Otto, Alex Bollen, Ingeborg M. Kooter, Nicole Moguilevsky, Nanna M. Sijtsema, Ron Wever, and SILS Other Research (FNWI)
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chemistry.chemical_classification ,Hypochlorous acid ,biology ,Stereochemistry ,Sulfonium ,Chromophore ,Biochemistry ,Cofactor ,Inorganic Chemistry ,chemistry.chemical_compound ,Enzyme ,chemistry ,Myeloperoxidase ,Pyridine ,biology.protein ,METIS-129334 ,Site-directed mutagenesis - Abstract
The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. more...
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- 1997
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26. Heme-Protein Interaction in Myeloperoxidase: Modification of Spectroscopic Properties and Catalytic Activity by Single Residue Mutation
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Alex Bollen, Rokus Renirie, Nicole Moguilevsky, René Floris, Ron Wever, Gerwin Puppels, Alain Jacquet, and SILS Other Research (FNWI)
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Hemeprotein ,biology ,Absorption spectroscopy ,Mutant ,General Chemistry ,Chromophore ,Biochemistry ,Catalysis ,law.invention ,chemistry.chemical_compound ,symbols.namesake ,Crystallography ,Colloid and Surface Chemistry ,chemistry ,law ,Myeloperoxidase ,symbols ,biology.protein ,sense organs ,Carboxylate ,Electron paramagnetic resonance ,Raman spectroscopy - Abstract
The optical absorbance spectrum of reduced myeloperoxidase shows an unusual red-shifted Soret band at 472 nm and an an α band at 636 nm. It has been speculated that this red-shift is due to interaction of the protein matrix with the chromophore. The carboxylate side chain of Glu242 is in close proximity of the prosthetic group of the enzyme, and we have examined the effect of the Glu242 to Gin mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation shifts the Soret band in the optical absorption spectrum of the reduced mutated enzyme from 472 to 458 nm. The EPR spectrum was hardly affected and was typical of a rhombic high-spin system (gx = 6.6, gy = 5.2). The alkaline pyridine hemochrome spectrum of the mutant was nearly identical to that of native myeloperoxidase. The resonance Raman spectrum, however, was drastically affected in the mutant. The symmetry-reducing effects were lifted by the mutation and the resonance Raman spectrum was indicative of an iron-porphyrin-like chromophore with a singlet va line at 1367 cm-1. The mutant enzyme was not able to peroxidize chloride to hypochlorous acid. We conclude that the interaction of residue Glu242 with the prosthetic group in native myeloperoxidase is partly responsible for the red-shifted Soret band in the optical spectrum and that this interaction is the origin of the symmetry-reducing effects in the resonance Raman spectrum of the native enzyme. This residue also plays a pivotal role in the ability of the enzyme to peroxidize chloride. more...
- Published
- 1995
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27. Pharmacological and Functional Characterisation of the Wild—Type and Site—Directed Mutants of the Human H1Histamine Receptor Stably Expressed in CHO Cells
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Alex Bollen, Henichart Jean-Pierre, J. Daliers, Francesca Varsalona, M. Noyer, Michel Gillard, J.-P. Guillaume, and Nicole Moguilevsky
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Molecular Sequence Data ,CHO Cells ,Histamine H1 receptor ,Biology ,Transfection ,Biochemistry ,chemistry.chemical_compound ,Histamine receptor ,Phosphatidylinositol Phosphates ,Histamine H2 receptor ,Cricetinae ,Animals ,Humans ,Amino Acid Sequence ,Receptors, Histamine H1 ,Cloning, Molecular ,Receptor ,Molecular Biology ,Pyrilamine ,Binding Sites ,Histamine binding ,Cell Biology ,Molecular biology ,Transmembrane protein ,Transmembrane domain ,chemistry ,Mutagenesis, Site-Directed ,Histamine - Abstract
A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine. more...
- Published
- 1995
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28. Exposure of endothelial cells to physiological levels of myeloperoxidase-modified LDL delays pericellular fibrinolysis
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Dany Brohée, Bassam Badran, Pierre Van Antwerpen, Michel Vanhaeverbeek, Jalil Daher, Paul Delrée, Nicole Moguilevsky, Martine Raes, Claude Remacle, Jean Ducobu, Karim Zouaoui Boudjeltia, Luc Vanhamme, and UCL - SST/ISV - Institut des sciences de la vie more...
- Subjects
Anatomy and Physiology ,medicine.medical_treatment ,Valvular Disease ,lcsh:Medicine ,Sciences biomédicales en général ,Coronary Artery Disease ,Cardiovascular ,Cardiovascular System ,Physiologie générale ,Blood plasma ,Molecular Cell Biology ,lcsh:Science ,Receptor ,Multidisciplinary ,biology ,Chemistry ,Fibrinolysis ,Venous Thromboembolism ,Hematology ,Angina ,Endothelial stem cell ,Lipoproteins, LDL ,Médecine interne ,Myeloperoxidase ,Medicine ,Tumor necrosis factor alpha ,lipids (amino acids, peptides, and proteins) ,Cellular Types ,Research Article ,medicine.medical_specialty ,Myocytes, Smooth Muscle ,Fibrin ,Vascular Biology ,Internal medicine ,medicine ,Human Umbilical Vein Endothelial Cells ,Humans ,Biology ,Peroxidase ,Tumor Necrosis Factor-alpha ,lcsh:R ,Cell Membrane ,Endothelial Cells ,medicine.disease ,Atherosclerosis ,Atheroma ,Endocrinology ,Immunology ,biology.protein ,lcsh:Q - Abstract
Blood fluidity is maintained by a delicate balance between coagulation and fibrinolysis. The endothelial cell surface is a key player in this equilibrium and cell surface disruptions can upset the balance. We investigated the role of pericellular myeloperoxidase oxidized LDLs (Mox-LDLs) in this balance., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published more...
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- 2011
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29. Regulation of macrophage function by human recombinant myeloperoxidase
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Alex Bollen, Stanley S. Lefkowitz, Kevin Mills, Austin Vaz, Nicole Moguilevsky, and Doris L. Lefkowitz
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Male ,medicine.medical_specialty ,Phagocytosis ,medicine.medical_treatment ,Immunology ,Alpha interferon ,Mice ,Azurophilic granule ,Adjuvants, Immunologic ,Internal medicine ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Immunology and Allergy ,Cells, Cultured ,Interferon alfa ,Peroxidase ,biology ,Tumor Necrosis Factor-alpha ,Macrophages ,Zymosan ,Macrophage Activation ,Molecular biology ,Recombinant Proteins ,Mice, Inbred C57BL ,Endocrinology ,Cell killing ,Cytokine ,Myeloperoxidase ,Interferon Type I ,biology.protein ,Tumor necrosis factor alpha ,Reactive Oxygen Species ,medicine.drug - Abstract
Myeloperoxidase is an enzyme which is found in the azurophilic granules of neutrophils and is associated with bactericidal, fungicidal, and tumoricidal activity. The present studies show that human recombinant myeloperoxidase (rec-MyPo) can regulate a number of macrophage (M phi) capacities and functions. Macrophages from mice exposed to rec-MyPo in vitro released reactive oxygen intermediates, tumor necrosis factor alpha (TNF alpha), and interferon alpha/beta (IFN alpha/beta). Enhanced target cell killing was also demonstrated with TNF alpha sensitive but not TNF alpha insensitive cells. Intravenous injection of rec-MyPo induced high titers of systemic TNF alpha and IFN alpha/beta. These results indicate that MyPo can function as an immunomodulator both in vitro and in vivo. Because of these actions, it is apparent that MyPo represents a previously unrecognized endogenous immunomodulator. more...
- Published
- 1993
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30. Production of authentic human proapolipoprotein A-I in Escherichia coli: Strategies for the removal of the amino-terminal methionine
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Alex Bollen, Kees Roobol, Pascal Gilles, J.-P. Guillaume, Francesca Varsalona, and Nicole Moguilevsky
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Enteropeptidase ,Signal peptide ,Recombinant Fusion Proteins ,Molecular Sequence Data ,DNA, Recombinant ,Bioengineering ,Biology ,Protein Engineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Aminopeptidase ,Methionine ,Escherichia coli ,medicine ,Humans ,Amino Acid Sequence ,Protein Precursors ,Apolipoproteins A ,Apolipoprotein A-I ,Base Sequence ,General Medicine ,Periplasmic space ,biology.organism_classification ,Molecular biology ,Enterobacteriaceae ,Fusion protein ,Enzymes ,Biochemistry ,Evaluation Studies as Topic ,Bacterial outer membrane ,Biotechnology - Abstract
Several methods were compared with respect to the production of authentic, N-terminal methionine-free proapolipoprotein A-I in engineered Escherichia coli bacteria. A first approach consisted of treating the purified methionylated recombinant protein with an amino-peptidase, purified from Aeromonas proteolytica. A second series of strategies was based on the construction of proapo A-I encoding cassettes carrying built-in recognition sites suitable for specific in vitro cleavage of the products with kallikrein and enterokinase, respectively. Along the same line, a fusion between ubiquitin and proapo A-I was produced in E. coli with the prospect to achieve post-purification cleavage with yeast ubiquitin hydrolase. Finally, proapo A-I was fused to the signal peptide of the bacterial outer membrane protein, OmpA, aiming at an in situ conversion to authentic proapo A-I during secretion to the bacterial periplasm. The data showed that, out of these five systems, the OmpA signal peptide system and, to a lesser extent, the one involving the fusion to ubiquitin were the most efficient in yielding authentic proapo A-I from engineered Escherichia coli. more...
- Published
- 1993
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31. Copper and myeloperoxidase-modified LDLs activate Nrf2 through different pathways of ros production in macrophages
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Laurine Mattart, Martine Raes, Alexandre Rousseau, Damien Calay, Pierre Van Antwerpen, Karim Zouaoui Boudjeltia, Cédric Delporte, Thierry Arnould, Vincent Nuyens, and Nicole Moguilevsky
- Subjects
NF-E2-Related Factor 2 ,Physiology ,Clinical Biochemistry ,Biology ,medicine.disease_cause ,Biochemistry ,Mice ,medicine ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,Peroxidase ,General Environmental Science ,Foam cell ,chemistry.chemical_classification ,Reactive oxygen species ,NADPH oxidase ,GCLM ,Macrophages ,Cell Biology ,Lipoproteins, LDL ,Oxidative Stress ,Cytosol ,chemistry ,Myeloperoxidase ,Leukocytes, Mononuclear ,biology.protein ,General Earth and Planetary Sciences ,lipids (amino acids, peptides, and proteins) ,Reactive Oxygen Species ,Copper ,Intracellular ,Oxidative stress - Abstract
Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages and in human peripheral blood monocyte-derived macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2 by RNAi. MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROS-producing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cytosolic PLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggest that results obtained until now with copper oxidized-LDLs should be carefully reevaluated, taking into consideration physiologically more relevant oxidized LDLs. © Mary Ann Liebert, Inc. more...
- Published
- 2010
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32. Glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase: glycosylation is required for optimal enzymatic activity
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Pierre, Van Antwerpen, Marie-Christine, Slomianny, Karim Zouaoui, Boudjeltia, Cedric, Delporte, Valegh, Faid, Damien, Calay, Alexandre, Rousseau, Nicole, Moguilevsky, Martine, Raes, Luc, Vanhamme, Paul G, Furtmüller, Christian, Obinger, Michel, Vanhaeverbeek, Jean, Nève, and Jean-Claude, Michalski more...
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Glycosylation ,Neutrophils ,Glycobiology and Extracellular Matrices ,CHO Cells ,Recombinant Proteins ,carbohydrates (lipids) ,Cricetulus ,Polysaccharides ,Cricetinae ,Animals ,Humans ,Protein Multimerization ,Protein Structure, Quaternary ,Peroxidase - Abstract
The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the three-dimensional structure of MPO. more...
- Published
- 2010
33. The vinyl-sulfonium bond in human myeloperoxidase: impact on compound I formation and reduction by halides and thiocyanate
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Martina Zederbauer, Bernadette Ganster, Paul G. Furtmüller, Nicole Moguilevsky, and Christian Obinger
- Subjects
Sulfonium ,Iodide ,Inorganic chemistry ,Biophysics ,Sulfonium Compounds ,Crystal structure ,Biochemistry ,Medicinal chemistry ,chemistry.chemical_compound ,Halogens ,Methionine ,Bromide ,Animals ,Humans ,Sulfones ,Molecular Biology ,Pyrrole ,Peroxidase ,chemistry.chemical_classification ,Thiocyanate ,Chemistry ,Order (ring theory) ,Valine ,Cell Biology ,Amino Acid Substitution ,Covalent bond ,Oxidation-Reduction ,Thiocyanates - Abstract
In human myeloperoxidase (MPO) the heme is covalently attached to the protein via two ester linkages and a unique sulfonium ion linkage between the sulfur atom of Met243 and the {beta}-carbon of the vinyl ring on pyrrole ring A. Here, we have investigated the variant Met243Val produced in Chinese hamster ovary cells in order to elucidate the role of the electron withdrawing sulfonium bond in compound I formation and reduction. Disruption of this MPO-typical bond causes a blue-shifted UV-vis spectrum and an increase in the heme flexibility. This had no impact on compound I formation mediated by hydrogen peroxide (2.2 x 10{sup 7} M{sup -1} s{sup -1} at pH 7.0 and 25 {sup o}C). Compared with wild-type recombinant MPO the cyanide association rate with ferric Met243Val was significantly enhanced as were also the calculated apparent bimolecular compound I reduction rates by iodide (>10{sup 8} M{sup -1} s{sup -1}) and thiocyanate (>10{sup 8} M{sup -1} s{sup -1}). By contrast, the overall chlorination and bromination activities were decreased by 98.1% and 87.4%, respectively, compared with the wild-type protein. Compound I reduction by chloride was slower than compound I decay to a compound II-like species (0.4 s{sup -1}), whereas compound I reduction bymore » bromide was about 10-times slower (1.3 x 10{sup 4} M{sup -1} s{sup -1}) than the wild-type rate. These findings are discussed with respect to the known crystal structure of MPO and its bromide complex as well as the known redox chemistry of its intermediates and substrates.« less more...
- Published
- 2007
34. Disruption of the aspartate to heme ester linkage in human myeloperoxidase: Impact on ligand binding, redox chemistry and interconversion of redox intermediates
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Martina, Zederbauer, Paul G, Furtmüller, Marzia, Bellei, Johanna, Stampler, Christa, Jakopitsch, Gianantonio, Battistuzzi, Nicole, Moguilevsky, and Christian, Obinger
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Aspartic Acid ,reduction potential compound I ,Spectrum Analysis ,Esters ,CHO Cells ,Heme ,Ligands ,Kinetics ,myeloperoxidase ,Cricetulus ,Cricetinae ,heme peroxidases ,Electrochemistry ,Animals ,Humans ,Oxidation-Reduction ,Peroxidase ,Protein Binding - Abstract
In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp(94) to heme ester bond decreased the one-electron reduction potential E'(0) [Fe(III)/Fe(II)] from 1 to -55 mV at pH 7.0 and 25 degrees C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp(94) to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands. more...
- Published
- 2007
35. Triggering of inflammatory response by myeloperoxidase-oxidized LDL
- Author
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Karim Zouaoui, Boudjeltia, Ilham, Legssyer, Pierre, Van Antwerpen, Roger Lema, Kisoka, Sajida, Babar, Nicole, Moguilevsky, Paul, Delree, Jean, Ducobu, Claude, Remacle, Michel, Vanhaeverbeek, and Dany, Brohee more...
- Subjects
Inflammation ,Lipoproteins, LDL ,Tumor Necrosis Factor-alpha ,Albumins ,Interleukin-8 ,Endothelial Cells ,Humans ,Inflammation Mediators ,Oxidation-Reduction ,Cells, Cultured ,Copper ,Monocytes ,Peroxidase - Abstract
The oxidation theory proposes that LDL oxidation is an early event in atherosclerosis and that oxidized LDL contributes to atherogenesis in triggering inflammation. In contrast to the copper-modified LDL, there are few studies using myeloperoxidase-modified LDL (Mox-LDL) as an inflammation inducer. Our aim is to test whether Mox-LDL could constitute a specific inducer of the inflammatory response. Albumin, which is the most abundant protein in plasma and which is present to an identical concentration of LDL in the intima, was used for comparison. The secretion of IL-8 by endothelial cells (Ea.hy926) and TNF-alpha by monocytes (THP-1) was measured in the cell medium after exposure of these cells to native LDL, native albumin, Mox-LDL, or Mox-albumin. We observed that Mox-LDL induced a 1.5- and 2-fold increase (ANOVA; P0.001) in IL-8 production at 100 microg/mL and 200 microg/mL, respectively. The incubation of THP-1 cells with Mox-LDL (100 microg/mL) increased the production of TNF-alpha 2-fold over the control. Native LDL, albumin, and Mox-albumin showed no effect in either cellular types. The myeloperoxidase-modified LDL increase in cytokine release by endothelial and monocyte cells and by firing both local and systemic inflammation could induce atherogenesis and its development. more...
- Published
- 2006
36. Sites of covalent attachment of CYP4 enzymes to heme: evidence for microheterogeneity of P450 heme orientation
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Jason T. Schuman, Nicole Moguilevsky, Kent L. Kunze, Matthew J. Cheesman, Allan E. Rettie, Brian R. Baer, Mariko Nakano, and Patricia A.P. Campbell
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chemistry.chemical_classification ,Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Hemeprotein ,Heme binding ,Cytochrome ,biology ,Chemistry ,Ligand ,Heme ,Biochemistry ,Conjugated protein ,Cofactor ,chemistry.chemical_compound ,Covalent bond ,biology.protein ,Aryl Hydrocarbon Hydroxylases ,Nuclear Magnetic Resonance, Biomolecular ,Chromatography, High Pressure Liquid - Abstract
Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality. Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems. more...
- Published
- 2005
37. Thiol-containing molecules interact with the myeloperoxidase/H2O2/chloride system to inhibit LDL oxidation
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Patrick Moreau, Ilham Legssyer, Michel Vanhaeverbeek, Sajida Babar, Jean Ducobu, Pierre Van Antwerpen, Nicole Moguilevsky, Jean Neve, and Karim Zouaoui Boudjeltia
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Captopril ,Apolipoprotein B ,Biophysics ,Oxidative phosphorylation ,Biochemistry ,Antioxidants ,Acetylcysteine ,chemistry.chemical_compound ,Chlorides ,medicine ,Sulfhydryl Compounds ,Molecular Biology ,Peroxidase ,chemistry.chemical_classification ,biology ,Lysine ,Cell Biology ,Glutathione ,Hydrogen Peroxide ,Flufenamic Acid ,Lipoproteins, LDL ,Flufenamic acid ,chemistry ,Myeloperoxidase ,Thiol ,biology.protein ,Oxidation-Reduction ,medicine.drug - Abstract
Oxidized low-density lipoproteins (LDL) accumulate in the vascular wall and promote a local inflammatory process contributing to the progression of atheromatous plaque. The key role of myeloperoxidase (MPO) in this process has been documented and the enzyme has been involved in the oxidative modification of apolipoprotein B-100 in the intima and at the surface of endothelial cells. As the inhibition of this last phenomenon could be of relevance in pharmacological interventions, thiol-containing molecules such as glutathione, captopril, and N-acetylcysteine (NAC) and its lysinate salt (NAL) were tested in this system and their properties were compared with those of flufenamic acid (control). This last compound already demonstrated an inhibition of the production of HOCl by MPO and a more intense inhibition of MPO activity than glutathione, NAC, NAL, and captopril. However, NAC and NAL inhibited the oxidative modification of LDL more intensively than captopril and glutathione whereas flufenamic acid had no comparable inhibiting effect. This could be related to the presence of LDL close to the catalytic site of the enzyme. NAC and NAL therefore appeared as the most efficient inhibitors probably as a consequence of their relatively small size. The relevance of such effects has to be documented by in vivo studies. more...
- Published
- 2005
38. Organic and inorganic substrates as probes for comparing native bovine lactoperoxidase and recombinant human myeloperoxidase
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Elena Maria Ghibaudi, Cristina Pacchiardo, Enzo Laurenti, Rosa Pia Ferrari, Nicole Moguilevsky, and Gianpaolo Suriano
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Biochemistry ,Adduct ,Substrate Specificity ,Inorganic Chemistry ,chemistry.chemical_compound ,Animals ,Lactoperoxidase ,Heme ,Peroxidase ,chemistry.chemical_classification ,Catechol ,biology ,Electron Spin Resonance Spectroscopy ,Substrate (chemistry) ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Kinetics ,Enzyme ,chemistry ,Myeloperoxidase ,Molecular Probes ,biology.protein ,Cattle - Abstract
The interaction of native bovine lactoperoxidase (nbLPO) with four substrates has been studied and compared with that of recombinant human myeloperoxidase (rhMPO). Kinetic, spectroscopic and binding parameters extrapolated for each enzyme–substrate adduct have been interpreted in the light of the structural data available for myeloperoxidase (X-ray structure) and lactoperoxidase (3D-model), respectively. The differences in the reactivity and affinity of nbLPO and rhMPO towards SCN−, catechol, dopamine and 3,4-dihydroxyphenylpropionic acid are here discussed and related to a different structure of the organic substrate access channel as well as to a different accessibility of the heme pocket in the two enzymes. more...
- Published
- 2003
39. Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands
- Author
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Franco Roviello, Nicola Grehan, Nicole Moguilevsky, Marc Mareel, José Carlos Machado, Raquel Seruca, Erik Bruyneel, Olivier De Wever, Maria Cristina Bordin, Ralph H. Hruban, Paulo Ferreira, Frances M. Richards, Carlos Caldas, David G. Huntsman, Carla Oliveira, Timothy R. Porter, Fátima Carneiro, and Gianpaolo Suriano more...
- Subjects
Adult ,Male ,Mutation, Missense ,CHO Cells ,medicine.disease_cause ,Germline ,CDH1 ,CDH1 germline missense mutations ,Germline mutation ,HDGC ,Stomach Neoplasms ,Cricetinae ,Genetics ,medicine ,gastric cancer ,E-cadherin ,E-cadherin protein ,Animals ,Humans ,Missense mutation ,Molecular Biology ,Genetics (clinical) ,Mutation ,biology ,Chinese hamster ovary cell ,General Medicine ,Middle Aged ,Cadherins ,medicine.disease ,biology.protein ,Carbohydrate Dehydrogenases ,Female ,Hereditary diffuse gastric cancer ,Carcinogenesis - Abstract
E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the two exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations. more...
- Published
- 2003
40. Major role for the carboxylic function of cetirizine and levocetirizine in their binding characteristics to human H1-histamine-receptors
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C. Van der Perren, R. Massingham, Michel Gillard, Nicole Moguilevsky, and Pierre Chatelain
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Immunology ,Carboxylic Acids ,Vascular permeability ,Histamine H1 receptor ,CHO Cells ,Pharmacology ,Levocetirizine ,Guinea pig ,Cricetinae ,medicine ,Animals ,Humans ,Cloning, Molecular ,Binding selectivity ,Cells, Cultured ,Chemistry ,Lysine ,Cell Membrane ,Stereoisomerism ,Smooth muscle contraction ,Receptor–ligand kinetics ,Cetirizine ,Kinetics ,Histamine H1 Antagonists ,Mutagenesis, Site-Directed ,medicine.drug - Abstract
H1 receptors are involved in smooth muscle contraction and increasing vascular permeability observed in allergic diseases. Cetirizine (ZyrtecTM) and levocetirizine (XyzalTM) are second generation antihistamines. As opposed to first generation drugs exemplified by chlorpheniramine, second generation drugs are less sedative, due to an improved H1 binding selectivity and reduced brain penetration [1]. Pharmacophores of H1 antagonists have been proposed based upon structure-activity relationships and from site-directed mutagenesis data obtained from guinea pig H1-receptors [2, 3]. Here, we provide additional elements to these models by investigating the role of Lys191 of the human H1-receptor in the binding kinetics of a series of close analogues of cetirizine. more...
- Published
- 2002
41. Binding characteristics of cetirizine and levocetirizine to human H(1) histamine receptors: contribution of Lys(191) and Thr(194)
- Author
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Pierre Chatelain, Michel Gillard, Christy Van der Perren, Nicole Moguilevsky, and R. Massingham
- Subjects
Threonine ,Stereochemistry ,Mepyramine ,Histamine H1 receptor ,CHO Cells ,Transfection ,Binding, Competitive ,Levocetirizine ,chemistry.chemical_compound ,Cricetinae ,medicine ,Animals ,Humans ,Receptors, Histamine H1 ,Cloning, Molecular ,Receptor ,Pharmacology ,Alanine ,Chemistry ,Lysine ,Cetirizine ,Receptor–ligand kinetics ,Kinetics ,Amino Acid Substitution ,Mutagenesis ,Molecular Medicine ,Stereoselectivity ,Histamine ,medicine.drug - Abstract
Competition experiments with [(3)H]mepyramine showed that cetirizine and its enantiomers, levocetirizine and (S)-cetirizine, bound with high affinity and stereoselectivity to human H(1) histamine receptors (K(i) values of 6, 3, and 100 nM, respectively). Cetirizine and levocetirizine were 600-fold more selective for H(1) receptors compared with a panel of receptors and channels. Binding results indicated that the interaction between cetirizine, its enantiomers, and histamine is compatible with a competitive behavior, in contrast with the noncompetitive profile of cetirizine and levocetirizine observed in isolated organs. Binding kinetics provided a suitable explanation for this observation, because levocetirizine dissociated from H(1) receptors with a half-time of 142 min; that of (S)-cetirizine was only 6 min, implying that the former could act as a pseudo-irreversible antagonist in functional studies. The carboxylic function of levocetirizine seemed responsible for its long dissociation time. Indeed, hydroxyl or methyl ester analogs dissociated more rapidly from H(1) receptors, with half-times of 31 min and 7 min, respectively. The importance of the carboxylic function of levocetirizine for the interaction with the H(1) receptor was further supported by the results from the mutation of Lys(191) to Ala(191). This mutation decreased the dissociation half-time of levocetirizine from 142 to 13 min and reduced its affinity from 3 to 12 nM, whereas the affinity and dissociation kinetics of hydroxyl and methyl ester analogs were hardly affected. The mutation of Thr(194) reduced the binding stereoselectivity by selectively enhancing the affinity of the distomer. more...
- Published
- 2002
42. Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase
- Author
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Shikiko Watanabe, Nicole Moguilevsky, Gianpaolo Suriano, Elena Maria Ghibaudi, Alex Bollen, and Rosa Pia Ferrari
- Subjects
Clinical Biochemistry ,Mutant ,Blotting, Western ,Glycine ,Pharmaceutical Science ,Glutamic Acid ,Enzyme-Linked Immunosorbent Assay ,CHO Cells ,Heme ,Biochemistry ,chemistry.chemical_compound ,Cricetinae ,Drug Discovery ,Animals ,Lactoperoxidase ,Site-directed mutagenesis ,Molecular Biology ,Polyacrylamide gel electrophoresis ,DNA Primers ,chemistry.chemical_classification ,Aspartic Acid ,Base Sequence ,Chemistry ,Chinese hamster ovary cell ,Organic Chemistry ,Valine ,Enzyme ,Amino Acid Substitution ,Covalent bond ,Mutation ,Molecular Medicine ,Cattle ,Electrophoresis, Polyacrylamide Gel - Abstract
In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed. more...
- Published
- 2001
43. Native and recombinant proteins to analyze auto-antibodies to myeloperoxidase in pauci-immune crescentic glomerulonephritis
- Author
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MM Boomsma, Wia W. Oost-Kort, Pieter Limburg, Jan Willem Cohen Tervaert, Cees G. M. Kallenberg, Nicole Moguilevsky, and Coen A. Stegeman
- Subjects
Male ,medicine.drug_class ,animal diseases ,Recombinant Fusion Proteins ,Immunology ,Enzyme-Linked Immunosorbent Assay ,Monoclonal antibody ,law.invention ,Antibodies, Antineutrophil Cytoplasmic ,Glomerulonephritis ,Antigen ,law ,Immunology and Allergy ,Medicine ,Rapidly progressive glomerulonephritis ,Humans ,cardiovascular diseases ,Fluorescent Antibody Technique, Indirect ,Anti-neutrophil cytoplasmic antibody ,Peroxidase ,biology ,business.industry ,Autoantibody ,Middle Aged ,medicine.disease ,Myeloperoxidase ,biology.protein ,Recombinant DNA ,Female ,Antibody ,business - Abstract
The prevalence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) in pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is dependent on the assay(s) used. We investigated the frequency of MPO-ANCA as detected by different assays for MPO-ANCA in a large cohort of patients with biopsy-proven pauci-immune NCGN. Sera from 121 consecutive untreated patients presenting with pauci-immune NCGN were tested for ANCA directed to proteinase-3 (PR3) at diagnosis. PR3-ANCA negative sera were tested by direct ELISA using recombinant or native MPO and by capture ELISA using two different specific monoclonal antibodies directed to MPO and three different antigenic sources. Sera from 80 relevant disease controls were tested to explore the specificity of the different assays. Thirty-eight out of 121 patients (31%) with pauci-immune NCGN did not have PR3-ANCA. Sufficient amounts of serum from 30 of these 38 PR3-ANCA negative patients were available for further testing. Recombinant and native MPO were recognized by similar numbers of sera in a direct ELISA (recombinant MPO: 93%, native MPO: 93%) and a capture ELISA (recombinant MPO: 77-87%, native MPO: 93%). Sera of patients with PR3-ANCA positive pauci-immune NCGN and disease controls were less frequently positive for MPO-ANCA in a capture ELISA (recombinant MPO: 3-7%, native MPO: 6-7%) than in a direct ELISA (recombinant MPO: 25%, native MPO: 13%). Both direct and capture ELISA assays using either native or recombinant MPO are sensitive techniques to detect MPO-ANCA in patients with pauci-immune NCGN. A capture ELISA performs better than a direct ELISA because it combines a higher specificity with a comparable sensitivity. Recombinant MPO is a good alternative for native MPO when used as antigen in a capture ELISA, but not when used in a direct ELISA because of lower specificity in this latter assay. more...
- Published
- 2001
44. Sequences (103-110) and (116-120) of the rat secretin receptor are implicated in secretin and VIP recognition
- Author
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Alex Bollen, Emmanuel Di Paolo, Magali Waelbroeck, Patrick Robberecht, and Nicole Moguilevsky
- Subjects
medicine.medical_specialty ,Receptors, Vasoactive Intestinal Polypeptide, Type I ,Recombinant Fusion Proteins ,Secretin receptor family ,Secretin family ,CHO Cells ,In Vitro Techniques ,General Biochemistry, Genetics and Molecular Biology ,Secretin ,Receptors, G-Protein-Coupled ,Receptors, Gastrointestinal Hormone ,History and Philosophy of Science ,Internal medicine ,Cricetinae ,medicine ,Animals ,Binding Sites ,Chemistry ,General Neuroscience ,Rats ,Kinetics ,Endocrinology ,Secretin receptor ,Receptors, Vasoactive Intestinal Peptide ,Adenylyl Cyclases ,Vasoactive Intestinal Peptide - Published
- 2001
45. Functional Structure of the Secretin Receptor
- Author
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Nicole Moguilevsky, Patrick Robberecht, and Magali Waelbroeck
- Subjects
Vasoactive intestinal peptide ,Secretin receptor family ,Secretin receptor ,Enteroendocrine cell ,Secretin family ,Signal transduction ,Receptor ,hormones, hormone substitutes, and hormone antagonists ,Cell biology ,Secretin - Abstract
Secretin is a 27 amino acid peptide secreted by specialised endocrine cells of the gut that regulates the pancreatic exocrine - and the liver bile secretions. Its role as a neuropeptide is likely but not yet proven. Secretin acts through interaction with a membrane receptor coupled to the Gαsprotein and thus stimulates cyclic AMP production. It belongs to a new subgroup of receptors named the GPCRB that includes - among others - the VIP, the PACAP, the Glucagon, the Glucagon like peptide 1, the Calcitonin and the Parathormone receptors. The expression in CHO cells of the wild type and of mutated receptors permits the identification of domains involved in ligand recognition, in signal transduction, in desensitisation and in receptor intemalisation. more...
- Published
- 2001
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46. Acquisition and Use of Myeloperoxidase in the Microbicidal Activity of Macrophages
- Author
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Laszlo Marodi, Christopher Tourney, Rita Kaposzta, Richard B. Johnston, and Nicole Moguilevsky
- Published
- 2000
- Full Text
- View/download PDF
47. Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds
- Author
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Henk L. Dekker, Alex Bollen, Nicole Moguilevsky, Ron Wever, Ingeborg M. Kooter, Nanna M. Sijtsema, Cees Otto, Damage and Repair in Cancer Development and Cancer Treatment (DARE), Faculty of Science and Technology, Medical Cell Biophysics, and SILS Other Research (FNWI) more...
- Subjects
Stereochemistry ,Sulfonium ,Mutant ,lactoperoxidase ,Glutamic Acid ,Heme ,Spectrum Analysis, Raman ,Biochemistry ,Residue (chemistry) ,chemistry.chemical_compound ,ester bond ,METIS-128421 ,IR-71491 ,Peroxidase ,Aspartic Acid ,Myeloperoxidase ,biology ,Lactoperoxidase ,Esters ,unusual heme ,Kinetics ,chemistry ,Covalent bond ,biology.protein ,Mutagenesis, Site-Directed - Abstract
The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I,M., Pierik, A.J., Merkx, M., Averill, B.A,, Moguilevsky, N., Bollen, A. Wever, R. (1997) J. Am. Chem. Sec. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found. more...
- Published
- 1999
48. Neutrophilic myeloperoxidase-macrophage interactions perpetuate chronic inflammation associated with experimental arthritis
- Author
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Stanley S. Lefkowitz, Alex Bollen, Suzanne R Graham, Joel D. Starnes, Monique P. Gelderman, Nicole Moguilevsky, Steven R. Fuhrmann, and Doris L. Lefkowitz
- Subjects
musculoskeletal diseases ,Pathology ,medicine.medical_specialty ,Systemic disease ,Erythema ,Neutrophils ,Streptococcus pyogenes ,medicine.medical_treatment ,Immunology ,Arthritis ,Inflammation ,Arthritis, Rheumatoid ,medicine ,Immunology and Allergy ,Animals ,Peroxidase ,biology ,business.industry ,Tumor Necrosis Factor-alpha ,Macrophages ,medicine.disease ,Rats ,Disease Models, Animal ,Cytokine ,Rats, Inbred Lew ,Rheumatoid arthritis ,Myeloperoxidase ,biology.protein ,Tumor necrosis factor alpha ,Female ,medicine.symptom ,business - Abstract
Rheumatoid arthritis is a systemic disease of unknown etiology. The purpose of this study was to elucidate an unrecognized interaction between neutrophilic myeloperoxidase (MPO) and macrophages (Mphi) which could perpetuate the inflammatory response associated with arthritis. A monoarticular arthritis was induced by intra-articular injection of group A streptococcus cell wall fragments (PG-APS) into the ankle joint of female Lewis rats. After swelling/erythema subsided, joints were reinjected with either recombinant MPO or enzymatically inactive MPO (iMPO). Joint measurements were made daily and arthritis was confirmed by histology. Neither iMPO nor MPO could initiate "clinical" arthritis; however, either form of the enzyme injected after PG-APS induced a dose-dependent increase in erythema and swelling. Mannans, which block the binding of MPO to Mo, ablated clinical symptoms. Also, the presence of tumor necrosis factor alpha was observed only in diseased joints using immunocytochemistry. more...
- Published
- 1999
49. HIV-1 infectivity and host range modification by cathepsin D present in human vaginal secretions
- Author
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Kamal El Messaoudi, Nicole Van Tieghem, Alex Bollen, Yvon Englert, Corinne Liesnard, Nicole Moguilevsky, and Lise Thiry
- Subjects
CD4-Positive T-Lymphocytes ,Receptors, CXCR4 ,Receptors, CCR5 ,Lymphocyte ,Immunology ,HIV Core Protein p24 ,Cathepsin D ,HIV Infections ,Biology ,Virus ,Microbiology ,chemistry.chemical_compound ,medicine ,Immunology and Allergy ,Humans ,Cells, Cultured ,Infectivity ,Cathepsin ,Infectious Diseases ,medicine.anatomical_structure ,chemistry ,Cell culture ,Vagina ,HIV-1 ,Female ,Pepstatin - Abstract
OBJECTIVE To investigate HIV-1 infectivity in the natural environment of vaginal secretions. DESIGN Vaginal wash samples collected from 14 healthy women were incubated in vitro with various HIV-1 strains for 10 min at 37 degrees C and then assayed for infectivity on primary lymphocyte cultures, or on CEM cells, or on CD4- ME180 cells derived from vaginal epithelium. METHODS HIV-1 infectivity was measured by early virus growth in the various host cells tested using a quantitative p24 assay and by the Karber procedure. RESULTS Preincubation of HIV-1(IIIB) with vaginal wash samples or 2 microg/ml cathepsin D increased the ability of the virus to grow in lymphocyte cultures. The vaginal wash effect was abolished by 5 microg/ml pepstatin A, an inhibitor of aspartyl proteases. Presence of precursor and mature forms of cathepsin D in vaginal wash was demonstrated after passage through a pepstatin A-agarose column. Median tissue culture infective doses of HIV-1(IIIB) and HIV-1(JRFL) strains were increased 14.4-fold and 18-fold, respectively, after preincubation in vaginal wash sample, and were increased by pretreatment with 2 microg/ml cathepsin D. When CD4 receptors of CEMss cells were blocked by OKT4a monoclonal antibody, the cells lost susceptibility to HIV-1 (IIIB), but supported the growth of virus pretreated with vaginal wash sample or cathepsin D. These treated viruses were able to initiate infection of CD4-ME180 epithelial cells, which were not receptive to untreated virus. ME180 cells were shown to possess the messenger of CXC-chemokine receptor-4. CONCLUSIONS Vaginal secretions may help HIV-1 transmission to women by increasing infectivity for CD4+ cells and allowing entrance into some CD4-epithelial cells. more...
- Published
- 1999
50. The sulfonium ion link in myeloperoxidase
- Author
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Nicole Moguilevsky, Henk L. Dekker, Ingeborg M. Kooter, Ron Wever, Cees Otto, Lars A. van der Veen, Alex Bollen, and SILS Other Research (FNWI)
- Subjects
biology ,Sulfonium ,Stereochemistry ,Lactoperoxidase ,Mutant ,Substituent ,Cell Biology ,Biochemistry ,Isotopic labeling ,chemistry.chemical_compound ,chemistry ,Covalent bond ,biology.protein ,Molecular Biology ,Heme ,Eosinophil peroxidase - Abstract
The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met243. Mutation of Met243 into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met243 mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the13CD3-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu242, it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins. more...
- Published
- 1999
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