Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency

Menegazzi, Renzo; Zabucchi, Giuliano; Zuccato, Patrizia; Cramer, Rita; Piccinini, Clara; Patriarca, Pierluigi

doi:10.1016/0022-1759(91)90393-t

Cited by 12 publications

(8 citation statements)

References 24 publications

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“…Nevertheless, using similarly puri fied proteins in the HVA-oxidation test system, pEPO showed the same pH profile as hEPO with high activity only between pH 10 and 11. thus being slightly different from that of liMPO. This is in contrast to the results obtained when eosinophil and neutrophil extracts were tested [23], yield ing high activity for EPO in the pi 1 range 7.4-10.5. An ex planation for the different results could be the presence of fluorescent elements in the extract, or that the conformation of the peroxidases is different in the unpurified form.…”

Section: Discussioncontrasting

confidence: 53%

“…However, pMPO has an absorbance maximum at 430 nm [27] and the possibility that the currently purified protein is pMPO could therefore be excluded. Peroxidase activity was measured as described by Menegazzi et al [23] and incubation buffers of various pH were tested. Interest ingly, we could detect only very low peroxidase activities for the purified peroxidases at pH 7.4, whereas activities were high at pH 10-11.…”

Section: Discussionmentioning

confidence: 99%

“…Peroxidase activity was determined using a homovanillic acid (I IVA) oxidation assay, modified from the method described by Menegazzi et al [23]. In the presence of hydrogen peroxide, the peroxidase catalyzes the oxidation of 11VA to a highly fluorescent compound II with an excitation maximum at 315 nm and an emission maximum at 425 nm [24].…”

Section: Peroxidase Activitymentioning

confidence: 99%

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Isolation and Characterization of Porcine Cationic Eosinophil Granule Proteins

Fornhem

Peterson²,

Alving³

1996

Int Arch Allergy Immunol

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The allergic pig can be used as a large-animal model for studies of allergic reactions in the airways and the role of eosinophils in such reactions. To measure the activation of eosinophils, the release of eosinophil-derived cationic proteins can be used. The purpose of this study was to isolate and characterize cationic proteins derived from porcine eosinophils. Pigs were infested with live Ascaris suum eggs to induce eosinophilia (≥ 40% of leukocytes). Blood was collected and leukocytes were prepared by dextran sedimentation. Granules were obtained from the homogenized leukocytes by ultracentrifugation and cationic proteins were extracted and separated by gel filtration, cation exchange and zinic affinity chromatography. Using these methods, three cationic proteins were isolated from pig granulocytes, two of which were shown to originate from the eosinophil. The proteins were characterized according to molecular weight, amino acid composition, N-terminal sequence, isoelectric point, peroxidase and ribonuclease activity and antigenicity. One eosinophil protein was identified as eosinophil peroxidase and the other showed great similarities with human eosinophil cationic protein. The third protein was not specific for eosinophils, and had no obvious equivalent in human granulocytes. The eosinophil-derived proteins may be useful in the studies of eosinophil activation, e.g. in late-phase asthmatic reactions, where the pig represents a new candidate model for large-animal allergy research.

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Section: Discussioncontrasting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Section: Peroxidase Activitymentioning

confidence: 99%

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Isolation and Characterization of Porcine Cationic Eosinophil Granule Proteins

Fornhem

Peterson²,

Alving³

1996

Int Arch Allergy Immunol

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“…A homovanillic acid (HVA) oxidation assay, as previously described (16), was used to assess EPO activity. Supernatant aliquots (100 l) were transferred to a 96-well plate and diluted with 100 l of 0.1 M glycine buffer (pH 10.5) containing 0.3% (vol/vol) hydrogen peroxide.…”

Section: Gel Electrophoresis and Western Blotting To Detect The Adhesmentioning

confidence: 99%

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation

Sawatzky

Kingham

Court

et al. 2002

American Journal of Physiology-Lung Cellular and Molecular Phys

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In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-α and interferon-γ intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C4. Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.

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“…Using purified horseradish peroxidase or polymorphonuclear leukocytes (PMNs), we compared the DCF method with the homovanillic acid (HVA) oxidation assay (Menegazzi et al 1991). A good correlation was found between these two assays (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Peroxidase deficiency of nickel-transformed hamster cells correlates with their increased resistance to cytotoxicity of peroxides

et al. 1996

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Using a procedure aimed at isolation of genes that are inactivated during nickel-induced carcinogenesis in Chinese hamster cells, a homolog of genes encoding human and mouse heme containing peroxidases has been cloned. Northern blot analysis of normal cultured fibroblasts and two nickel-transformed cell lines confirmed that this gene was expressed in normal but not in transformed cells. Nickel-transformed cells also tested negative for peroxidase activity using a sensitive fluorescence assay. Cultured embryo cells or fibroblasts that express peroxidase activity and their nickel-transformed peroxidase-deficient counterparts were employed to investigate the role of peroxidase-catalyzed processes in cytotoxicity induced by tert-butyl hydroperoxide or cumene hydroperoxide. It has been found that peroxidase-deficient cells were significantly more resistant to cytotoxic effect of these compounds suggesting that cytotoxic effect of hydroperoxides may be mediated in part by free radicals generated in the course of peroxidase-catalyzed reactions.

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Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency

Cited by 12 publications

References 24 publications

Isolation and Characterization of Porcine Cationic Eosinophil Granule Proteins

Isolation and Characterization of Porcine Cationic Eosinophil Granule Proteins

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation

Peroxidase deficiency of nickel-transformed hamster cells correlates with their increased resistance to cytotoxicity of peroxides

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