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Biomedical subjects

D Meredith

Publications and source records attributed to D Meredith.

At least 19 recordsLinked to original sources
PubMed2000

Characterisation of human monocarboxylate transporter 4 substantiates its role in lactic acid efflux from skeletal muscle.

Monocarboxylate transporter (MCT) 4 is the major monocarboxylate transporter isoform present in white skeletal muscle and is responsible for the efflux of lactic acid produced by glycolysis. Here we report the characterisation of MCT4 expressed in Xenopus oocytes. The protein was correctly targeted to the plasma membrane and rates of substrate transport were determined from the rate of intracellular acidification monitored with the pH-sensitive dye 2', 7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In order to validate the technique, the kinetics of monocarboxylate transport were measured in oocytes expressing MCT1. Km values determined for L-lactate, D-lactate and pyruvate of 4.4, > 60 and 2.1 mM, respectively, were similar to those determined previously in tumour cells. Comparison of the time course of [14C]lactate accumulation with the rate of intracellular acidification monitored with BCECF suggests that the latter reflects pH changes close to the plasma membrane associated with transport, whilst the former may include diffusion-limited movement of lactate into the bulk cytosol. Km values of MCT4 for these substrates were found to be 28, 519 and 153 mM, respectively, and for a range of other monocarboxylates values were at least an order of magnitude higher than for MCT1. Vmax values appeared to be similar for all substrates. K0.5 values of MCT4 (determined at 30 mM L-lactate) for inhibition by alpha-cyano-4-hydroxycinnamate (991 microM), phloretin (41 microM), 5-nitro-2-(3-phenylpropylamino)benzoate (240 microM), p-chloromercuribenzene sulphonate (21 microM) and 3-isobutyl-1-methylxanthine (970 microM, partial inhibition) were also substantially higher than for MCT1. No inhibition of MCT4 by 2 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonate was observed. The properties of MCT4 are consistent with published data on giant sarcolemmal vesicles in which MCT4 is the dominant MCT isoform, and are appropriate for the proposed role of MCT4 in mediating the efflux from the cell of glycolytically derived lactic acid but not pyruvate.

Animals↗
PubMed2000

Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb.

The goals of the work reported here were (i) to identify distinct GABA(A)/benzodiazepine receptors in the rat hippocampus and olfactory bulb using receptor binding assays, and (ii) to determine the affinities and selectivities of benzodiazepine receptor ligands from structurally diverse chemical families at each site identified. These studies were aided by the use of software AFFINITY ANALYSIS SYSTEM, developed in our laboratory for analysis of receptor binding data that allows the determination of receptor heterogeneity using non-selective radioligands. Saturation binding assays using [3H]RO15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a]-[1,4]benzodiazepine-3-carboxylate) revealed two binding sites in each of these two tissues. The higher affinity site corresponds to alpha(5) subunit-containing GABA(A) receptor and the lower affinity site to a combination of alpha(1), alpha(2), and alpha(3) subunit-containing receptors. These results should be useful in the challenging task of identifying the various functional GABA(A) receptors in the central nervous system, and in providing a link between receptor affinities and in vivo activities of the GABA(A)/benzodiazepine receptor ligands studied.

Animals↗
PubMed2000

Distinct transcriptional and functional properties of the R transactivator gene orf50 of the transforming herpesvirus saimiri strain C488.

The transformation-associated region of herpesvirus saimiri strains is variable, whereas other parts of the virus genome are highly conserved. However, we observed considerable interstrain sequence divergence of the early viral regulatory orf50 gene, which encodes the R transactivator, a homolog of Epstein-Barr virus BRLF1. The orf50 gene of strain C488 was transcribed at low abundance during lytic infection, whereas antisense transcripts were simultaneously expressed at high levels. A spliced variant, orf50a, was detectable by RT-PCR and RNase protection assays in stimulated C488-transformed, nonpermissive human T cells. In contrast to strain A11, the short, unspliced orf50b form of C488 displayed complete transactivation capability on the orf6 and orf57 promoters. In summary, there are unexpected structural and functional differences between the orf50 genes of herpesvirus saimiri strains, which differ in their capability to transform human T lymphocytes.

Amino Acid Sequence↗
PubMed2000

Structure and function of eukaryotic peptide transporters.

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.

Amino Acid Sequence↗
PubMed2000

Characterization of benzodiazepine receptors in the cerebellum.

1. The goals of the work reported here were to further characterize benzodiazepine/GABA(A) (BDZR) receptor heterogeneity in the cerebellum and to measure the affinities and selectivities of structurally diverse benzodiazepines at each site identified. 2. Five chemical families were included in these studies. These were 1,4-benzodiazepines (flunitrazepam), imidazobenzodiazepines (RO15-1788 and RO15-4513 and RO16-6028), beta-carbolines (Abecarnil) and pyrazoloquinolines (CGS 8216, CGS 9895 and CGS 9896). 3. Saturation and competition binding assays were combined with powerful data analysis software developed in our laboratory. Among the capabilities of this software is the identification of multiple binding sites for a cold ligand using a non-selective labeled ligand that binds with equal, but high, affinity to all the binding sites 4. Saturation binding assays using either [3H]-RO15-1788 or [3H]-RO15-4513 revealed only one apparent binding site, with a higher affinity for RO15-4513 than for RO15-1788. However, using [3H]-RO15-4513 for the competition binding studies in the cerebellum, together with our data analysis software, led to the identification of two distinct binding sites with equal densities for the diverse benzodiazepines studied. 5. In rat cerebellum one of the sites identified corresponds to GABA(A) receptors exhibiting alpha1 subunit pharmacology and the other to GABA(A) receptors exhibiting alpha6 subunit pharmacology. In general, the diverse families of BDZR ligands studied had much lower affinities for the alpha6 containing receptors.

Animals↗
PubMed2000

Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1.

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.

Amino Acids↗
PubMed1998

4-aminomethylbenzoic acid is a non-translocated competitive inhibitor of the epithelial peptide transporter PepT1.

1. 4-Aminomethylbenzoic acid, a molecule which mimics the special configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus Laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles. 2. This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1-exp ressing oocytes and indirectly by its failure to trans-stimulate labelle d peptide efflux through PepT1 in oocytes and in renal membrane vessicle s. 3. However 4-aminiomethylbenzoic acid does reverse trans-stimulation through expressed PepT1 of labelled peptid efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4-aminomethyl benzoic acid competitively binding to the external surface of PepT1. 4. 4-Aminomethylbenzoic acid (the first molecule discovered to be a non-translocated competitive inhibitor of proton-coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.

4-Aminobenzoic Acid↗
PubMed1998

Peptide mimics as substrates for the intestinal peptide transporter.

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated. 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter. These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.

Aniline Compounds↗
PubMed1998

Fulminant desquamative interstitial pneumonitis.

A previously well, 48-year-old female presented with a short history of respiratory distress and fever for which no cause could be found. Open lung biopsy was not contributory. She died within nine days of presentation of hypoxia and multi-organ failure. Post mortem examination revealed the pattern of desquamative interstitial pneumonitis.

Female↗
PubMed1996

Both the H13 gene product and 4F2 antigen are involved in the induction of system y+ cationic amino-acid transport following activation of human peripheral blood mononuclear cells (PBM).

Prior transfection with antisense oligonucleotides to the H13 and 4F2 hc genes, singly or in combination, was found to inhibit phytohaemagglutinin-induced activation of cationic amino-acid transport system y+ in human peripheral blood mononuclear cells (mostly circulating lymphocytes). These effects on system y+ function or expression mean that 4F2 hc cannot only be the molecular basis of system y+L (Fei, Y.-J., Prasad, P.D., Leibach, F.H. and Ganapathy, V. (1995) Biochemistry 34, 8744-8751).

Amino Acids↗
PubMed1995

Dipeptide transport characteristics of the apical membrane of rat lung type II pneumocytes.

The transport of a hydrolysis-resistant dipeptide, D-phenylalanyl-L-alanine (D-Phe-L-Ala), has been studied by high-performance liquid chromatography in rat lung epithelial cells and apical membrane vesicles. Time-dependent uptake of D-Phe-L-Ala into isolated type II pneumocytes was shown. Uptake was saturable, and Michaelis-Menten kinetics were fitted to the data and gave an apparent Michaelis constant (Km) of 3.4 mM and a maximum velocity (Vmax) of 7.0 nmol.mg protein-1.min-1. However, known peptide transport inhibitors unexpectedly increased intracellular D-Phe-L-Ala concentration when initial rates of peptide uptake were studied. Apical (brush-border) membrane vesicles prepared from rat lung also showed time- and concentration-dependent influx of D-Phe-L-Ala (apparent Km 2.0 mM, Vmax 0.53 nmol.mg protein-1.min-1). Influx of this neutral dipeptide into the vesicles was shown to be both electrogenic and stimulated by an inwardly directed proton gradient. Influx was inhibitable by mercuric chloride and by the amino acid residue modifying compounds N-acetylimidazole and diethylpyrocarbonate. These findings strongly suggest the presence of a proton-coupled peptide transport protein in the apical surface of the type II cell. This transporter may play a role in lung homeostasis.

Animals↗
PubMed1995

Patient selection criteria for home i.v. therapies: from A to Z.

Home intravenous therapy is a fast-growing industry, but it may not be for everyone: several medical and safety criteria must be met to ensure effective patient care. Home care agencies should examine each patient situation closely before deciding to implement a home i.v. program.

Caregivers↗
PubMed1994

Tripeptide transport in rat lung.

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

Amino Acid Sequence↗
PubMed1994

Semiquantum chaos.

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Journal Article↗
PubMed1991

Abnormal erythrocyte choline transport in patients with chronic renal failure.

1. Erythrocyte choline transport has been studied in nine patients on maintenance haemodialysis for chronic renal failure, six patients on continuous ambulatory peritoneal dialysis, 31 patients with renal transplants and in nine normal control subjects. 2. The mean maximum rate of choline influx (Vmax., measured at an extracellular choline concentration of 250 mumol/l) was 66.7 (SD 14.1) mumol h-1 l-1 cells in patients on haemodialysis, 87.8 (SD 18.5) mumol h-1 l-1 cells in patients on continuous ambulatory peritoneal dialysis and 30.5 (SD 4.9) mumol h-1 l-1 cells in control subjects. The increase in choline flux in patients on haemodialysis and patients on continuous ambulatory peritoneal dialysis compared with control subjects was highly significant (P less than 0.001). 3. Renal transplant patients showed variable values for the Vmax. of choline influx (range 17.7-71.7 mumol h-1 l-1 cells). The values showed a significant negative correlation with creatinine clearance and this correlation correctly extrapolated to the maximum choline flux in normal subjects and in patients on dialysis. 4. The kinetics of choline transport have been studied in erythrocytes of patients on haemodialysis and control subjects in 'zero-trans' conditions after depletion of intracellular choline. The mean Vmax. in these conditions was 38.4 (SD 4.6) mumol h-1 l-1 cells in patients on haemodialysis compared with 14.2 (SD 3.7) mumol h-1 l-1 cells in control subjects. The mean Km under 'zero-trans' conditions was 19.4 (SD 2.4) mumol/l in patients on haemodialysis and 7.4 (SD 1.4) mumol/l in control subjects. These differences were significant (P less than 0.001).

Biological Transport↗