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B A Luxon

Publications and source records attributed to B A Luxon.

At least 19 recordsLinked to original sources
PubMed1995

Nuclear magnetic resonance solution structure of an undecanucleotide duplex with a complementary thymidine base opposite a 10R adduct derived from trans addition of a deoxyadenosine N6-amino group to (-)-(7R,8S,9R,10S)-7,8-dihydroxy- 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The solution structure of a modified undecamer duplex containing (-)-(7R,8S,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene covalently bonded through trans ring opening at C10 of the epoxide by the N6-amino group of deoxyadenosine (dA) was studied. This diol epoxide 1 diastereomer has the benzylic 7-hydroxyl group and the epoxide oxygen cis. The modified nucleotide residue has R chirality at C10 of the hydrocarbon (10R adduct). The undecamer duplex d(C1G2G3T4C5A*6C7G8A9G10G11).d(C12C13T14C15G16T17G18A19C2 0C21G22) has a complementary T opposite the modified dA (dA*6 is the modified dA). Exchangeable and nonexchangeable proton assignments were made using 2D TOCSY, NOESY, and water/NOESY NMR spectroscopy. The hybrid complete relaxation matrix program MORASS was used to generate NOESY distance constraints for iterative refinement using distance-restrained molecular dynamics calculations. The refined structure showed the hydrocarbon intercalated from the major groove between dA*6-T17 and dC5-dG18 base pairs. The modified dA*6 was in the normal anti configuration and showed Watson-Crick base pairing to T17 opposite. The chemical shifts of the hydrocarbon protons and the unusual shifts of sugar protons were accounted for by the intercalated orientation of the hydrocarbon.

Base Composition↗
PubMed1995

Is there facilitated uptake of fatty acids by the liver? Interpretation and analysis of experimental data.

Uptake of hydrophobic organic anions that are extensively bound to serum proteins has been a controversial issue for over 30 years. It is known that steady-state uptake is lower in the presence of binding proteins, but it is much higher than predicted on the basis of protein-ligand binding equilibrium. Several theories have been postulated to account for this observation. Recent work has shown how binding proteins are capable of enhancing the uptake rate of long-chain fatty acids by decreasing the diffusional resistance of the unstirred fluid layer. The enhanced transport via codiffusion is especially important for tightly bound ligands like long-chain fatty acids. Whether this model accounts for all experimental data or whether hepatocytes facilitate the uptake of protein-bound ligands, by for example mediating the protein-ligand dissociation rate, is not clear. We review the published reports to gain an understanding into the potential mechanism for the extraction of long-chain fatty acids. Understanding the uptake mechanism of these important metabolic substrates is vitally important in determining their overall utilization in a variety of clinical disorders as diverse as gallstones, obesity, and atherosclerosis.

Animals↗
PubMed1995

Intralobular zonal heterogeneity and hepatic indicator dilution curves.

Conventional interpretation of hepatic indicator dilution curves rests on the assumption, among others, that every hepatocyte operates with the same rate constants. When this assumption is false, owing to intralobular zonal variation in surface-to-volume ratios and/or to zonal differences in permeability, the apparent rate constants recoverable from outflow transients are wrong estimates of average liver performance. We develop the theoretical basis for this conclusion and illustrate by example how it can confuse the interpretation of experimental data. The analysis proceeds from vascular and extracellular reference curves recorded from perfused rat livers and from a simple model of intralobular architecture in which highly arborized periportal sinusoids have a larger surface-to-volume ratio than the less-branched vasculature around the central vein. The experimental data and the model, applied to a wide range of hypothetical solutes, are used to compare the true average rate constants for uptake, efflux, and intracellular removal with the apparent values recoverable from outflow curves. When zonal differences in surface-to-volume ratios are the sole source of heterogeneity, the wrong estimates prove of little practical importance. By contrast, assigning larger regional variations in permeability leads to substantial errors. The confusion arising from such errors may be qualitative as well as quantitative. The presence of heterogeneity and thus the risk of interpretive error appears unrecognizable from outflow curves.

Animals↗
PubMed1995

Hepatic transport of rose bengal by perfused rabbit liver: the effect of albumin binding on the unidirectional rate constants.

The effect of albumin on the unidirectional rate constants for the hepatic transport of rose bengal was studied using perfused rabbit livers. Rabbit livers were perfused in a recirculating system with albumin concentrations between 10 and 600 microM and the disappearance of I-125 rose bengal following a bolus injection was recorded. A distributed model of hepatic transport was used to estimate the rate constants for influx into cells, efflux from cells to plasma and biliary excretion. When the rate constants were corrected for albumin binding, the influx, but not the efflux or excretion constant, was a steeply rising function of the perfusate albumin concentration. The result that the influx but not efflux constant is albumin dependent suggests that the phenomenon is not due to slow diffusion across an unstirred fluid layer or to nonequilibrium binding within such a layer. The possibility that the albumin-dependent influx is due to a direct exchange of rose bengal between albumin and a membrane carrier protein is also considered. The independence of the efflux constant and the albumin concentration makes this hypothesis less tenable. However, these data are consistent with the facilitation of albumin-ligand dissociation by liver cells. This analysis represents the first reduction to practice of distributed modeling of disappearance curves. The fact that the excretion constant is independent of the perfusate albumin concentration serves to validate the model that has previously only been considered on theoretical grounds.

Animals↗
PubMed1993

2D 1H and 31P NMR spectra and distorted A-DNA-like duplex structure of a phosphorodithioate oligonucleotide.

Assignment of the 1H and 31P NMR spectra of a phosphorodithioate modified oligonucleotide decamer duplex, d(CGCTTpS2-AAGCG)2 (10-mer-S; a site of dithioate substitution is designated with the symbols pS2-), was achieved by two-dimensional homonuclear TOCSY, NOESY and 1H-31P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. In contrast to the parent palindromic decamer sequence (1) which has been shown to exist entirely in the duplex B-DNA conformation under comparable conditions (100 mM KCl), the dithiophosphate analogue forms a hairpin loop. However, the duplex form of the dithioate oligonucleotide can be stabilized at lower temperatures, higher salt and strand concentration. The solution structure of the decamer duplex was calculated by an iterative hybrid relaxation matrix method (MORASS) combined with 2D NOESY-distance restrained molecular dynamics. These backbone modified compounds, potentially attractive antisense oligonucleotide agents, are often assumed to possess similar structure as the parent nucleic acid complex. Importantly, the refined structure of the phosphorodithioate duplex shows a significant deviation from the parent unmodified, phosphoryl duplex. An overall bend and unwinding in the phosphorodithioate duplex is observed. The structural distortion of the phosphorodithioate duplex was confirmed by comparison of helicoidal parameters and groove dimensions. Especially, the helical twists of the phosphorodithioate decamer deviate significantly from the parent phosphoryl decamer. The minor groove width of phosphorodithioate duplex 10-mer-S varies between 8.4 and 13.3 A which is much wider than those of the parent phosphoryl decamer d(CGCTTAAGCG)2 (4.2 approximately 9.4 A). The larger minor groove width of 10-mer-S duplex contributes to the unwinding of the backbone and indicates that the duplex has an overall A-DNA-like conformation in the region surrounding the dithiophosphate modification.

Base Sequence↗
PubMed1992

Hepatic uptake of 3,5,3'-triiodothyronine: electrochemical driving forces.

We used the multiple indicator dilution technique to assess the electrochemical forces driving uptake of 3,5,3'-triiodo-L-thyronine (T3) across the basolateral plasma membrane in the single-pass perfused rat liver. With the use of 4 g/dl albumin solutions, the influx and efflux clearances were 0.020 +/- 0.005 and 0.0049 +/- 0.0017 (SE) ml.s-1.g liver-1, respectively, indicating that the total T3 concentration at equilibrium should be about four times greater in cytoplasm than in plasma. However, when the influx and efflux clearances were divided by the unbound (free) T3 concentration in the perfusate and cytosol, they were not different (3.76 +/- 0.26 vs. 4.30 +/- 0.38 ml.s-1.g liver-1), indicating that the uptake process does not generate a gradient of unbound T3 across the plasma membrane. To further test whether T3 uptake is driven by the electrical potential difference across the plasma membrane, liver cells were depolarized by isosmotic replacement of perfusate chloride with gluconate. There was no effect on uptake or efflux. To test whether uptake is coupled to influx of sodium, perfusate sodium was replaced with choline. Although there was a modest decline in both the influx and efflux clearances, there was no change in their ratio, as would be expected for sodium-coupled active transport. These results indicate that uptake of T3 across the basolateral hepatocyte membrane occurs by passive diffusion. We found no evidence to support concentrative, active transport by either electrogenic or sodium-coupled mechanisms.

Animals↗
PubMed1992

On the design and interpretation of experiments to elucidate albumin-dependent hepatic uptake.

The liver's apparently anomalous extraction of organic anions tightly bound to albumin continues to provoke controversy and confusion. Decisive experiments have proved difficult to design, and mathematical models have usually been constructed to defend one or another putative mechanism to the exclusion of others. To stimulate more decisive experiments and as an aid to interpreting those already reported, we discuss a general formulation of the problem that predicts the clearance pattern to be expected when facilitated dissociation and codiffusion are joint determinants of the uptake flux. The results provide an approach to modeling the various mechanisms by which the concentration of bound ligand at the cell surface could be a driving force for uptake. Further we present new calculations to clarify the interpretation of net ligand clearance when the removal of free ligand is the result of bidirectional fluxes into and out of an unstirred sink. Applied to a previously published comparison of the uptake performances of hepatocytes and polyethylene, the new calculations support the inference that facilitated dissociation of albumin-palmitate complexes occurs at or near the hepatocyte surface.

Animals↗
PubMed1992

A new method for quantitating intracellular transport: application to the thyroid hormone 3,5,3'-triiodothyronine.

After entering cells from plasma, molecules must permeate through the cytoplasm before they can be metabolized or excreted. If sufficiently slow, cytoplasmic transport may determine the overall rate of cellular elimination at steady state. Cytoplasmic transport of amphipathic molecules should be particularly slow because of extensive binding to intracellular membranes and proteins. Traditional transport models assume that molecules become instantly available for metabolism and canalicular excretion after entering the cell and thus cannot be used to assess cytoplasmic transport. We therefore extended the traditional multiple-indicator dilution (MID) method of Goresky to explicitly incorporate cytoplasmic transport and used the resulting model to estimate the rate constant for cytoplasmic transport of the amphipathic thyroid hormone 3,5,3'-triiodothyronine (T3). We chose T3 because control studies indicated that it is neither metabolized nor excreted during the brief period of an MID experiment (40-90 s). The traditional MID model was unable to account for the data unless we postulated rapid metabolism or excretion of T3. In contrast, the new diffusion MID model fit the data closely without this false assumption and gave values for the influx and efflux rate constants that agreed with previously published data. The half-time for equilibration of T3 across the cytoplasm of the hepatocyte averaged 50 s. This corresponds to an effective cytoplasmic diffusion constant of 3.1 x 10(-8) cm2/s, which is > 100 times slower than expected for free T3 in water. Our results indicate that cytoplasmic transport of this model amphipathic compound is much slower than membrane transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Only free bile acid drives ileal absorption of taurocholate.

We compared the intestinal absorption of monomeric taurocholate bound to albumin with the absorption of the free form. In the presence of albumin the apparent uptake coefficient is about three times greater than that in its absence. This result resembles the one reported previously for the liver. In the gut, however, this phenomenon is attributable to the diffusive resistance of an unstirred intervillous fluid layer, whereas in the liver this explanation has been excluded. We conclude that only free taurocholate engages the bile acid transport system in ileal mucosa, whereas in the liver bile acid uptake is driven by albumin-taurocholate complexes, as well as by free taurocholate.

Albumins↗
PubMed1986

Palmitate uptake by hepatocyte monolayers. Effect of albumin binding.

The uptake of 14C-palmitate by rat liver cell monolayers is depressed by binding of the fatty acid to albumin. When the uptake flux is divided by the concentration of free palmitate in the bathing medium, however, the resulting clearance is approximately 14 times greater in the presence of albumin than in its absence. These findings are not accounted for by the different diffusion rates of free and bound palmitate across an unstirred fluid layer, nor attributable to nonequilibrium binding. Instead we argue that the most plausible explanation is accelerated dissociation of albumin-palmitate complexes mediated by the cell surface--an interpretation that also explains the uptake kinetics of other albumin-bound organic anions by perfused rat liver.

Adsorption↗
PubMed1985

Hepatic transport and binding of rose bengal in the presence of albumin and gamma globulin.

Gamma globulin and albumin are compared with respect to their effects on the hepatic transport of rose bengal and with respect to the rates and affinities with which they bind this dye. The apparent intrinsic clearance of rose bengal is greater in the presence of albumin than in the presence of gamma globulin, and this difference increases with the protein concentration. Because the binding affinities of these proteins also differ, however, it cannot be concluded decisively that the mechanisms of dye removal are distinct. For both proteins the binding reaction rates as measured by stopped-flow spectrophotometry are much faster than the rate of convection along the sinusoid or the rate of removal of free dye by liver cells. The transport data and the binding rate constants are the basis for an extended theoretical model developed and analyzed in an accompanying report.

Animals↗
PubMed1985

Effects of unstirred Disse fluid, nonequilibrium binding, and surface-mediated dissociation on hepatic removal of albumin-bound organic anions.

Proceeding from the observation that organic anions bound to albumin have hepatic extraction fractions that are unexpectedly high, we have studied a distributed model that accounts for this phenomenon by invoking sites on the cell surface that catalyze the dissociation of albumin-anion complexes. The present report extends this model to include nonequilibrium binding and rate-limiting diffusion of bound anion to the cell surface. Simulation analysis of the extended model provides an unambiguous basis for interpreting the apparent intrinsic clearance of free anion. The model is fully consistent with the transport data that we obtained with rose bengal [Am. J. Physiol. 248 (Gastrointest. Liver Physiol. 11): G702-G708, 1985] but is suited to estimating only selected components of the unknown parameter vector. Uncertainties inherent in an alternate approach are discussed and illustrated.

Animals↗
PubMed1985

Effect of a transported ligand on the binding of albumin to rat liver cells.

Organic anions destined for hepatic uptake often bind to albumin in the circulation. Because albumin binds to liver cells but is not transported, we suggest that sites on the hepatocyte surface catalyze the dissociation of albumin-anion complexes, thus making more free anion available for transport than would otherwise occur. To learn whether liver cells distinguish between free albumin and albumin-anion complexes, we measured the binding of 125I-albumin to isolated rat hepatocytes in the presence and absence of rose bengal, a transported anion that binds extensively to albumin. Albumin binding to hepatocytes is reported as the albumin space corrected for extracellular fluid (14C-inulin space). Corrected albumin spaces are 2.95 and 2.83 microliter/mg cell protein with and without rose bengal, respectively. The mean difference and its 95% confidence interval computed from four comparisons in each of six rats is 0.12 +/- 0.67 microliter/mg cell protein. Inulin space is 32% of the uncorrected albumin space. Thus the affinities of albumin-rose bengal complexes and of free albumin for the hepatocyte surface differ by at most 28%. Accordingly, free albumin can compete with albumin-rose bengal complexes for cell surface sites, impairing the surface-mediated generation of free rose bengal for uptake. This finding explains the otherwise paradoxical observation that adding albumin to liver perfusate inhibits the uptake of rose bengal even when sufficient albumin is already present to bind 99.9% of this dye.

Animals↗
PubMed1984

Determining hepatic transport kinetics by mathematical modeling.

Three approaches to the measurement of hepatic transport kinetics by mathematical modeling are briefly reviewed. The so-called lumped, two-compartment model and the single-pass multiple-indicator dilution method enjoy a long history of wide application, but each is subject to important errors inherent in their underlying assumptions. An understanding of the reasons for these errors, as revealed by simulation analysis, suggests a third approach that can substantially improve the previous difficulties.

Animals↗
PubMed1983

Phosphorus-31 nuclear magnetic resonance of ethidium complexes with ribonucleic acid model systems and phenylalanine-accepting transfer ribonucleic acid.

The temperature dependence of the 31P NMR spectra of the ethidium complexes with poly(A) X oligo(U) and the 31P spectra of phenylalanine tRNA (yeast) in various molar ratios of ethidium ion (Et) are presented. In the poly(A) X oligo(U) X Et complex, a new peak about 2.0 ppm downfield from the double-helix peak appears. We have assigned this peak to phosphates perturbed by ethidium. The chemical shift of this peak is consistent with the intercalation mode of binding and provides additional support for our hypothesis that 31P shifts are sensitive probes of phosphate ester conformations. The main effect of ethidium on the 31P spectra of tRNAPhe is the broadening of several of the scattered signals. These scattered signals are associated with phosphates involved in tertiary interactions. We propose that these broadened signals arise from phosphates near the Et binding site.

Ethidium↗
PubMed1983

Analyzing tracer disappearance curves to study hepatic transport kinetics.

This paper is devoted to a discussion of recent developments in the compartmental analysis of hepatic transport, especially the interpretation of tracer disappearance curves recorded from the reservoir of an isolated perfused liver preparation. The emphasis is on the advantages that this approach enjoys over other mathematical models, and a critical review of alternative methods is therefore included. The mathematical equations are largely suppressed, however, having appeared elsewhere in detail.

Animals↗