A method to measure permeability of red blood cell membrane to water and solutes using intrinsic fluorescence.

BACKGROUND: Designing effective cryopreservation procedures for cells requires knowledge of permeability of cell membrane to water and solutes. To determine cell membrane permeability, one needs to measure the rate of cell volume changes in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which complicates the use of traditional methods. Preservation of RBCs from umbilical cord blood for neonatal transfusions is currently broadly discussed in the literature, but data on osmotic permeability of cord RBCs is controversial. Therefore, alternative methods to determine osmotic membrane permeability of these cells are warranted. We describe a technique to measure rapid changes in RBC volume through changes in the intensity of RBC autofluorescence. METHODS: To induce osmotically-driven changes in RBC volume, we rapidly mixed human RBCs with anisotonic solutions in a stopped-flow spectroscopy system and the intensity of intrinsic RBC fluorescence was measured. RESULTS: We found that change in RBC volume cause a proportional change in the intensity of RBC autofluorescence. This phenomenon occurs due to the self-quenching of RBC hemoglobin autofluorescence at high intracellular concentrations. CONCLUSIONS: This novel method to determine osmotic permeability of RBCs overcomes the limitations of traditional techniques and has numerous clinical applications.

The discovery and development of selective 3-fluoro-4-aryloxyallylamine inhibitors of the amine oxidase activity of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1).

A new class of 3-fluoroallyl amine-based SSAO/VAP-1 inhibitors is reported. These compounds have excellent selectivity over diamine oxidase, MAO-A and MAO-B. Synthesis and SAR studies leading to compound 28 (PXS-4159A) are reported. The pharmacokinetic profile of 28 in the rat, together with activity in a murine model of lung inflammation are also disclosed.

On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware.

Plastic consumables, used universally in bioscience laboratories, are presumed inert with respect to bioassay outcomes. However, it is clear that many pipette tips, microfuge tubes, and other plastic disposables leach bioactive compounds into assay solutions, profoundly affecting data and experimental interpretation. In this paper we discuss the nature and sources of leachates and review several examples of compromised bioassay data that speak to the probable widespread nature of this largely unrecognised source of error. Strategies for minimizing leachate interferences are discussed.

Characterization of the in vitro binding and inhibition kinetics of primary amine oxidase/vascular adhesion protein-1 by glucosamine.

BACKGROUND: Primary-amine oxidase (PrAO) catalyzes the oxidative deamination of endogenous and exogenous primary amines and also functions, in some tissues, as an inflammation-inducible endothelial factor, known as vascular adhesion protein-1. VAP-1 mediates the slow rolling and adhesion of lymphocytes to endothelial cells in a number of inflammatory conditions, including inflammation of the synovium. METHODS: Glucosamine binding to the enzyme was assessed spectrofluorometrically and the kinetics of inhibition of PrAO were determined spectrophotometrically through the use of direct or coupled assays, in the presence of different substrates. RESULTS: Glucosamine is not a substrate for PrAO, but acts as a time-dependent inhibitor of PrAO activity, displaying mixed inhibition kinetics. The observed inhibition and binding were augmented in the presence of H(2)O(2). CONCLUSIONS: Significant in vitro effects on PrAO require glucosamine in the millimolar concentration range and it is not clear at this stage whether a low but persistent level of PrAO inhibition might contribute to the anti-arthritic response. GENERAL SIGNIFICANCE: This work was aimed at characterizing the interactions of PrAO/VAP-1 with glucosamine, a widely used "over-the-counter" supplement for the treatment of osteoarthritis.

An improved approach to steady-state analysis of monoamine oxidases.

The search for new monoamine oxidase inhibitors aims to identify potential lead compounds that are more potent and selective than current drugs for use in treating a variety of neuropsychiatric and neurodegenerative conditions. An integral part of this process is a kinetic examination of monoamine oxidases in the presence of the inhibitor, to determine potency and selectivity and to obtain information on mechanism. To date, kinetic data obtained with a probe substrate have been analysed by fitting to the Michaelis-Menten equation which describes a unireactant process in which velocity is related to substrate concentration in a rectangular hyperbolic manner. In this study, we present evidence that monoamine oxidase activity is often not adequately described by this approach. We outline a novel equation strategy that takes account of substrate and inhibitor binding to oxidised and reduced enzyme forms, and quantifies differences between substrates and inhibitors in this regard. When combined with plate reader-based experimental techniques that allow large numbers of substrate and inhibitor concentrations to be used, and the global nonlinear regression facilities of GraphPad Prism software, this straightforward approach allows more appropriate analyses of monoamine oxidase by non-experts than has previously been possible.

From caffeine to fish waste: amine compounds present in food and drugs and their interactions with primary amine oxidase.

Tissue bound primary amine oxidase (PrAO) and its circulating plasma-soluble form are involved, through their catalytic activity, in important cellular roles, including the adhesion of lymphocytes to endothelial cells during various inflammatory conditions, the regulation of cell growth and maturation, extracellular matrix deposition and maturation and glucose transport. PrAO catalyses the oxidative deamination of several xenobiotics and has been linked to vascular toxicity, due to the generation of cytotoxic aldehydes. In this study, a series of amines and aldehydes contained in food and drugs were tested via a high-throughput assay as potential substrates or inhibitors of bovine plasma PrAO. Although none of the compounds analyzed were found to be substrates for the enzyme, a series of molecules, including caffeine, the antidiabetics phenformin and tolbutamide and the antimicrobial pentamidine, were identified as PrAO inhibitors. Although the inhibition observed was in the millimolar and micromolar range, these data show that further work will be necessary to elucidate whether the interaction of ingested biogenic or xenobiotic amines with PrAO might adversely affect its biological roles.

On the formation and nature of the imidazoline I2 binding site on human monoamine oxidase-B.

An allosteric binding site with high affinity for imidazoline I(2) ligands has been proposed to exist on monoamine oxidase-B (MAO-B). However, enzyme inhibition only occurs at ligand concentrations far higher than are required to saturate this site. We here confirm previous reports that inactivation of recombinant human MAO-B with tranylcypromine results in the formation of a high affinity I(2) site on the enzyme, measured as an increase in binding of [(3)H]2-BFI. Incubation of MAO-B with 2-phenylethylamine, an endogenous trace amine and MAO-B substrate, resulted in a progressive loss of enzyme activity, increased enzyme mass, distinct spectral changes and, as was observed with tranylcypromine, a parallel increase in high affinity binding of [(3)H]2-BFI. Kinetic studies of the mechanism by which 2-BFI inhibits MAO-B activity suggested binding of 2-BFI, at micromolar concentrations, to a site distinct from the active site on at least two forms of the pure enzyme, probably corresponding to oxidised and reduced enzyme states. Studies with mutant enzymes revealed a pattern of changes consistent with binding of 2-BFI to the substrate entrance channel of human MAO-B. Structural data confirm that high affinity binding of I(2) ligands occurs within the entrance channel of inactive enzyme, while lower affinity binding at the same location in catalytically active enzyme results in mixed inhibition of MAO-B activity. High affinity I(2) sites may form in vivo due to inactivation of a portion of MAO-B during amine oxidation, while the low affinity I(2) site on active enzyme is a target for novel MAO-B inhibitor drugs.

Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction of L-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since D-lysine, L-lysine ethyl ester and epsilon-acetyl-L-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified.