Biomedical subjects

D Barron

Publications and source records attributed to D Barron.

At least 19 recordsLinked to original sources

PubMed2005

In vitro biological properties of flavonoid conjugates found in vivo.

For some flavonoids such as quercetin, isoflavones and catechins, the pathways of absorption and metabolism are now reasonably well characterised and understood. By definition, for biological activity of flavonoids to be manifest, the target tissue, which includes the blood and vascular system, must respond to the form(s) of flavonoid that it encounters. Bioavailability studies have shown that the circulating form of most flavonoids is as conjugates, with a few notable exceptions. There have been several recent papers on the in vitro biological properties of conjugates that have been found in vivo. This paper reviews the properties of these conjugates. Most of the information currently available is on quercetin glucuronides, but also on isoflavone and catechin conjugates. In addition to the biological properties of the conjugates, the partition coefficients and methods of synthesis are also presented.

Biological Availability↗

PubMed2005

The incidence of S. aureus bacteraemia in acute hospitals of the Mid-Western Area, Ireland, 2002-2004.

Concerns about healthcare-associated infections and the global crisis in antimicrobial resistance has combined to accentuate the fears around so-called "superbugs". In Ireland there is no single agreed indicator regarded as a true measure of the level of methicillin resistant Staphylococcus aureus (MRSA) in hospitals. The objective of this study was to compare two crude measures of MRSA--the percentage of bacteraemia caused by MRSA and the incidence rate (per 1000 bed days used) of MRSA bacteraemia in six acute hospitals. We examined all blood cultures positive for S. aureus (methicillin sensitive and resistant) from 2002 to 2004 in the Health Service Executive (HSE) Mid-Western Area of Ireland. Hospital In-Patient Enquiry (HIPE) data was used to determine monthly in-patient bed days used. Of 245 patient episodes of bacteraemia, 119 were MRSA. The trends in the percentage of isolates that were MRSA and the incidence rate calculated were compared. The incidence rate appears to be a more reliable and robust indicator of MRSA in hospitals than the percentage. Despite many difficulties in interpreting indicators of MRSA they should not preclude the regular publication of data at least at regional level in Ireland.

Acute Disease↗

PubMed2003

Multiple flavonoid-binding sites within multidrug resistance protein MRP1.

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N3-[alpha-32P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C4 (LTC4) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N3-[alpha-32P]ADP trapping, weakened inhibition of LTC4 transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.

Adenosine Diphosphate↗

PubMed2002

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Multidrug resistance phenotype mediated by the P-glycoprotein-like transporter in Leishmania: a search for reversal agents.

Abstract: Protozoan parasites are responsible for important diseases that threaten the lives of nearly one-quarter of the human population world-wide. Among them, leishmaniasis has become the second cause of death, mainly due to the emergence of parasite resistance to conventional drugs. P-glycoprotein (Pgp)-like transporters overexpression is a very efficient mechanism to reduce the intracellular accumulation of many drugs in cancer cells and parasitic protozoans including Plasmodium and Leishmania, thus conferring a multidrug resistance (MDR) phenotype. Therefore, there is a great clinical interest in developing inhibitors of these transporters to overcome such a resistance. Pgps are active pumps belonging to the ATP-binding cassette (ABC) superfamily of proteins, and consist of two homologous halves, each containing a transmembrane domain (TMD) involved in drug efflux, and a cytosolic nucleotide-binding domain (NBD) responsible for ATP binding and hydrolysis. Most conventional cancer MDR modulators interact with the drug-binding sites on the TMDs of Pgps, but they are also usually transported and the required concentrations for a permanent inhibition produce subsequent side-effects that hamper their clinical use. Besides, they only poorly modulate the resistance in protozoan parasites. We review here a rational strategy developed to overcome the MDR phenotype in Leishmania, consisting in: i) the selection of an MDR Leishmania tropica line that overexpresses a Pgp-like transporter; ii) the use of their cytosolic NBDs as new pharmacological targets; iii) the search of new natural compounds that revert the MDR phenotype in Leishmania by binding to the TMDs; iv) the combination of subdoses of the above selected modulators directed to both targets in the transporter, NBDs and TMDs, to accumulate their reversal effects while diminishing their toxicity. In this way, we have reverted the MDR phenotype in Leishmania, including the resistance to the most promising new antileishmania agents, the alkyl-lysophospholipids. This approach might be extrapolated to be used in other eukaryotic cells.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2001

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

C-Isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance.

Previous studies have shown that flavones bind to P-glycoprotein (Pgp) with higher affinity than isoflavones, flavanones, and glycosylated derivatives. In the present work, a series of C- or O-substituted hydrophobic derivatives of chrysin were synthesized to further investigate structural requirements of the A ring toward Pgp modulation. Increasing hydrophobicity at either position 6, 8, or 7 increased the affinity of in vitro binding to a purified cytosolic domain of Pgp, but only benzyl and 3,3-dimethylallyl C-substitution produced a high maximal quenching of the protein intrinsic fluorescence. Inhibition of membrane Pgp within leukemic cells, characterized by intracellular drug accumulation, was specifically produced by isoprenylated derivatives, with 8-(3,3-dimethylallyl)chrysin being even more efficient than the commonly used cyclosporin A.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Natural and synthetic benzophenones: interaction with the cytosolic binding domain of P-glycoprotein.

A benzophenone glycoside has been isolated from Davallia solida. Its structure was elucidated by chemical and spectral means as 4-O-beta-D-glucopyranosyl-2,6,4'-trihydroxybenzophenone. It bound with moderate affinity to the purified C-terminal cytosolic domain of P-glycoprotein, but the binding affinity was 6- to 10-fold increased for its aglycone derivative and other related benzophenones.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin.

The position of conjugation of the flavonoid quercetin dramatically affects biological activity in vitro, therefore it is important to determine the exact nature of the plasma metabolites. In the present study, we have used various methods (HPLC with diode array detection, LCMS, chemical and enzymic synthesis of authentic conjugates and specific enzymic hydrolysis) to show that quercetin glucosides are not present in plasma of human subjects 1.5 h after consumption of onions (a rich source of flavonoid glucosides). All four individuals had similar qualitative profiles of metabolites. The major circulating compounds in the plasma after 1.5 h are identified as quercetin-3-glucuronide, 3'-methylquercetin-3-glucuronide and quercetin-3'-sulfate. The existence of substitutions in the B and/or C ring of plasma quercetin metabolites suggests that these conjugates will each have very different biological activities.

Adult↗

PubMed2001

Right ventricular distension alters monophasic action potential duration during pulmonary arterial occlusion in anaesthetised lambs: evidence for arrhythmogenic right ventricular mechanoelectrical feedback.

Abnormal loading and distension of the right ventricle may induce arrhythmia through the process of mechanoelectrical feedback. Nonetheless, the electrophysiological effects of right ventricular distension are ill-defined and the mechanisms which underpin mechanoelectrical feedback in the right ventricle are unknown. We examined the effects of changes in right ventricular load (complete occlusion of both caval veins or the main pulmonary artery) in 14 anaesthetised lambs, instrumented with right ventricular surface electrodes and strain gauges for recording monophasic action potential and segment length, and an integrated conductance and micromanometer-tipped catheter for measurement of right ventricular pressure and volume. Caval occlusion did not alter right ventricular segment length and monophasic action potential duration. By contrast, pulmonary arterial occlusion increased the segment length and decreased the monophasic action potential duration at 25, 50 and 70% repolarisation by 29 +/- 6, 22 +/- 4 and 17 +/- 3 ms, respectively (all P < 0.01). Of the 42 pulmonary arterial occlusions, 38 were associated with early afterdepolarisations (EADs) which increased progressively in magnitude as the occlusion was maintained until, in 32, overt arrhythmia was observed. By contrast, none of the four occlusions in which EADs were not observed resulted in arrhythmia. As a result, the proportion of occlusions which resulted in arrhythmia were greater in those associated with EADs than in those which were not (P = 0.002). Right ventricular distension alters the pattern of repolarisation, precipitates early afterdepolarisations and results in a variety of ventricular arrhythmia, including ventricular tachycardia.

Action Potentials↗

PubMed2001

High-affinity binding of silybin derivatives to the nucleotide-binding domain of a Leishmania tropica P-glycoprotein-like transporter and chemosensitization of a multidrug-resistant parasite to daunomycin.

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Prenylated xanthones as potential P-glycoprotein modulators.

Dimethylallyl (DMA) derivatives of a naturally occurring xanthone (decussatin 1) were prepared. Their activity as potential P-glycoprotein inhibitors was monitored by affinity of direct binding and compared to that of corresponding DMA-flavones. Both classes of compounds exhibited the same structure-activity relationships. Decreasing polarity enhanced the binding affinity for the P-glycoprotein C-terminal cytosolic domain since DMA derivatives were more active, but unsubstituted hydroxyl group close to the carbonyl was required for efficient activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae.

The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive. Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter.

ATP-Binding Cassette Transporters↗

PubMed2000

The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of P-glycoprotein.

A new series of potential flavonoidic modulators of P-glycoprotein activity has been prepared. The flavanolignan silybin was first oxidised to dehydrosilybin and then C-alkylated with either prenyl or geranyl bromide. The resulting isoprenoid dehydrosilybins were shown to display high in vitro affinities for direct binding to P-glycoprotein, which ranged them among the best flavonoids ever tested.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Video teleconferencing with realistic simulation for medical education.

This report describes how realistic patient simulation can be used with video teleconferencing to conduct long-distance clinical case discussions with realistic re-enactments of critical events. By observing what appears to be a real procedure unfolding in real time, it is intended that audience members will better learn and appreciate the lessons from conferences. A commercially available mannequin simulator and video teleconferencing technology were used in nine sessions between a free-standing simulation center and different conference sites throughout the U.S. Transmission was via high-speed telephone lines. In each conference, a clinical scenario was simulated on a screen. Audience members asked questions of a live simulated "patient" and family and later advised the care team on routine treatments and management of urgent clinical problems that arose during management of the mannequin simulator in a highly realistic clinical setting. Ninety-eight percent of respondents from one audience of 150 (response rate 60%) judged the quality of the presentation as "very good or excellent." In response to the statement that "the educational value of the presentation was much greater than that of a standard case conference," 95% scored 4 or 5 on a five-point Likert scale (where 5 is highest agreement). While all conferences were conducted successfully, there were instances of technical challenge in using teleconferencing technology. Technical information about the teleconferencing system and scenario preparation, contingency planning for failures, and other details of using this new teaching modality are described. Although audiences were enthusiastic in their response to this approach to clinical case conferences, further study is needed to assess the added value of interactive simulation for education compared to standard conference formats.

Education, Medical↗

PubMed2000

Phenylpropanoids from Umbilicus pendulinus.

Phytochemical investigation of the leaves of Umbilicus pendulinus afforded in addition to 2-O-caffeoyl malate, isoquercitrin and Z-venusol, the new isomer E-venusol. Special NMR experiments were carried out to elucidate the configuration of the two latter compounds.

Caffeic Acids↗

PubMed1999

Correlation between the affinity of flavonoids binding to the cytosolic site of Leishmania tropica multidrug transporter and their efficiency to revert parasite resistance to daunomycin.

The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid analogue RU 486, and different classes of flavonoids with the following efficiency: flavone > flavanone > isoflavone > glucorhamnosyl-flavone > chromone. The affinity for flavones was dependent on the presence of hydroxyl groups at positions 5 and 3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position 8. When flow cytometry was used to measure daunomycin accumulation in a MDR L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentration or apigenin at higher concentration, but neither with the glucorhamnosyl derivative rutin nor with the isoflavone genistein. The in vivo reversing effect of dimethylallyl-kaempferide was correlated to a high inhibition of MDR cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein.

A hexahistidine-tagged C-terminal nucleotide-binding domain (H6-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated to fluorescence resonance-energy transfer. H6-NBD2 also bound all flavonoids known to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells, with similar affinities and relative efficiencies. Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4'-methoxy 3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of H6-NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by preincubation with ATP, or partly displaced upon ATP addition. Interestingly, kaempferide was also able to partly prevent the binding of the antiprogestin RU 486 to a hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, which occurred at the ATP site and extended to the vicinal steroid-interacting hydrophobic region, was completely prevented or displaced by kaempferide. All results indicate that flavonoids constitute a new class of modulators with bifunctional interactions at vicinal ATP-binding site and steroid-interacting region within a cytosolic domain of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗