Biomedical subjects

E M Wright

Publications and source records attributed to E M Wright.

At least 73 records · Page 4Linked to original sources

PubMed1999

Fetal ultrasound biometry: 2. Abdomen and femur length reference values.

OBJECTIVE: To create reliable reference ranges and calculate Z scores for fetal abdomen and femur ultrasound biometry using a large sample size which is evenly distributed from 12 to 42 weeks of pregnancy. DESIGN: A prospective, cross-sectional study. SETTING: Obstetric clinics (outpatient and delivery units) at the University Hospital of Zurich. SAMPLE: The study data were obtained from 6557 pregnant women. METHODS: Only the first ultrasound examination between 12 and 42 weeks of each fetus with certainly established gestational age was used for analysis. No exclusions were made on the grounds of small-for-date birthweight, prematurity or other events several weeks after the examination. Separate regression models were fitted to estimate the mean and standard deviation at each gestational age for each parameter. RESULTS: A total of 5807 mean abdominal diameters and abdominal circumferences were derived from fetal transverse and anterio-posterior fetal abdominal diameter measurements. Fetal femur length was measured in 5860 instances. The charts, tables and regression formulae of the biometrical measurements are presented. A comparison of our charts with others showed no significant difference. Only Merz's centiles for abdominal biometry were lower and for femur length higher than ours. An application to calculate Z scores was developed using Excel (Microsoft Corporation, USA); the macros are presented in detail in the Figure 6 footnote. CONCLUSIONS: We have presented centile charts, tables and formulae for fetal abdominal diameter and circumference and femur length derived from a large and minimally selected sample size in a carefully designed cross-sectional study. Complete tables and regression formulae to calculate reference ranges and Z scores are presented to use in computer-aided evaluation of fetal ultrasound biometry.

Abdomen↗

PubMed1999

Calculating reference intervals for laboratory measurements.

Reference intervals are used in laboratory medicine to detect measurements which are extreme, possibly abnormal. Methods of estimating reference intervals and age-specific reference intervals (where the measurement is dependent on a covariate, typically age) are reviewed. The issues of calculating confidence bands, determining appropriate sample sizes and assessing goodness-of-fit are discussed.

Adult↗

PubMed1998

Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with alpha-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 +/- 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 +/- 2 transmembrane helices, connexin 50 is a hexamer containing 24 +/- 3 helices, the major intrinsic protein is a tetramer containing 24 +/- 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 +/- 2 helices.

Animals↗

PubMed1998

Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1.

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.

Alanine↗

PubMed1998

Conformational changes couple Na+ and glucose transport.

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady-state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady-state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Alkylation↗

PubMed1998

The human Na+-glucose cotransporter is a molecular water pump.

1. The human Na+-glucose cotransporter (hSGLT1) was expressed in Xenopus laevis oocytes. The transport activity, given by the Na+ current, was monitored as a clamp current and the concomitant flux of water followed optically as the change in oocyte volume. 2. When glucose was added to the bathing solution there was an abrupt increase in clamp current and an immediate swelling of the oocyte. The transmembrane transport of two Na+ ions and one sugar molecule was coupled, within the protein itself, to the influx of 210 water molecules. 3. This stoichiometry was constant and independent of the external parameters: Na+ concentrations, sugar concentrations, transmembrane voltages, temperature and osmotic gradients. 4. The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein from the downhill fluxes of Na+ and sugar to the uphill transport of water, indicative of secondary active transport of water. 5. Unstirred layer effects were ruled out on the basis of experiments on oocytes treated with gramicidin or other ionophores. Na+ currents maintained by ionophores did not lead to any initial water movements. 6. The finding of a molecular water pump allows for new models of cellular water transport which include coupling between ion and water fluxes at the protein level; the hSGLT1 could account for almost half the daily reuptake of water from the small intestine.

Animals↗

PubMed1998

Relationships between Na+/glucose cotransporter (SGLT1) currents and fluxes.

The relationships between currents generated by the rabbit Na+/glucose cotransporter (SGLT1) and the fluxes of Na+ and sugar were investigated using Xenopus laevis oocytes expressing SGLT1. In individual voltage-clamped oocytes we measured: (i) the current evoked by 10 mM alpha MG and the 22Na+ uptake at 10 mM Na+; (ii) the currents evoked by 50 to 500 microM [14C]alpha MG and the [14C]alpha MG uptakes at 100 mM Na+; and (iii) phlorizin-sensitive leak currents in the absence of sugar and 22Na+ uptakes at 10 mM Na+. We demonstrate that the SGLT1 leak currents are Na+ currents, and that the sugar-evoked currents are directly proportional to both alpha MG and Na+ uptakes. The Na+/alpha MG coupling coefficients were estimated to be 1.6 at -70 mV and 1.9 at -110 mV. This suggests that the rabbit SGLT1 Na+/alpha MG stoichiometry for sugar uptake is 2 under fully saturating, zero-trans conditions. Coupling coefficients of less than 2 are expected under nonsaturating conditions due to uncoupled Na+ fluxes (slippage). The similarity between the Na+ Hill coefficients and the coupling coefficients suggests strong cooperativity between the two Na+ binding sites.

Animals↗

PubMed1998

How to construct 'normal ranges' for fetal variables.

A method to estimate reference intervals which depend on gestational age is described. The approach is based on standard statistical techniques, so implementation should be straightforward with most statistical packages. The reference limits are defined by simple formulae which may be plotted as smooth curves. The method is illustrated with analyses of three real datasets arising from different aspects of fetal monitoring.

Cephalometry↗

PubMed1998

I. Glucose galactose malabsorption.

Glucose Galactose Malabsorption is a genetic disorder caused by a defect in glucose and galactose transport across the intestinal brush border. Normally, lactose in milk is broken down into glucose and galactose by lactase, an ectoenzyme on the brush border, and the hexoses are transported into the cell by the Na+-glucose cotransporter SGLT1. The mutations causing the defect in sugar transport have been identified in patients from 33 kindreds, and functional studies have established how these mutations cause the disease.

Animals↗

PubMed1998

Functional expression of the rabbit intestinal Na+/L-proline cotransporter (IMINO system) in Xenopus laevis oocytes.

Proline absorption across small intestine takes place mainly through a Na+-dependent cotransporter localized at the brush border membrane of the enterocyte named IMINO system. It transports L-proline and 4-OH-proline but not L-alanine, neither cationic nor anionic amino acids. The present work demonstrates the functional expression of this transporter in Xenopus laevis oocytes by mRNA microinjection and radiotracer uptake techniques. Poly (A)+-RNA was isolated from rabbit jejunal mucosa and injected into oocytes. Five days after the injection, results showed 1.5 fold stimulation of 50 microM 3H-proline uptake by the injected oocytes when compared to the non injected oocytes uptake. Poly (A)+-RNA was sized fractionated and fractions were injected again. Increase on Na+-dependent L-proline uptake was obtained with a mRNA fraction between 2,4 and 4,4 kb, which was used to construct a cDNA library. The library was sequentially divided and cRNAs injected into oocytes in order to screen for an increment on the signal. A subdivision containing around 2,000 colonies was found to augment L-proline uptake 25 fold over the non injected oocytes uptake. This cRNA pool was used to further characterize the transporter. Results showed that in the absence of Na+ there was no L-proline uptake, 2 mM 4-OH-L-proline completely inhibited 50 microM proline uptake and there was no 50 microM alanine uptake. In summary, these results demonstrate the expression of the rabbit small intestine IMINO transporter in Xenopus laevis oocytes and support the next steps in the isolation of the clone.

Alanine↗

PubMed1998

Structure and function of the Na+/glucose cotransporter.

Cotransporters are a major class of membrane transport proteins that are responsible for the accumulation of nutrients, neurotransmitters, osmolytes and ions in cells from bacteria to man. The energy for solute accumulation comes from the proton and/or sodium electrochemical gradients that exist across cell membranes. A major problem in biology is how transport is coupled to these electrochemical potential gradients. The primary example of this class of membrane proteins is the intestinal brush border Na+/glucose cotransporter (SGLT1), first described by Bob Crane in 1960. Over 35 members of the SGLT1 gene family have been identified in animal cells, yeast and bacteria, and all share a common core structure of 13 transmembrane (TM) helices. Electrophysiological techniques have been used to examine the function of several family members, chimeras and mutants expressed in heterologous systems such as Xenopus laevis oocytes. These have revealed that cotransporters are multi-functional proteins: they are responsible for 1). uncoupled passive Na+ transport (Na+ uniport); 2). down-hill water transport in the absence of substrate; 3). Na+/substrate cotransport; and 4). Na+/substrate/water cotransport. The sugar binding and translocation pathway is formed by 4 TM helices near the C-terminal of the protein, helices 10-13. We propose that the N-terminal domains of SGLT1 are responsible for Na+ binding and/or translocation, and that Na+/glucose cotransport results from interactions between the N- and C-terminal domains of the protein.

Animals↗

PubMed1997

Simplified estimation of age-specific reference intervals for skewed data.

Age-specific reference intervals are commonly used in medical screening and clinical practice, where interest lies in the detection of extreme values. Many different statistical approaches have been published on this topic. The advantages of a parametric method are that they necessarily produce smooth centile curves, the entire density is estimated and an explicit formula is available for the centiles. The method proposed here is a simplified version of a recent approach proposed by Royston and Wright. Basic transformations of the data and multiple regression techniques are combined to model the mean, standard deviation and skewness. Using these simple tools, which are implemented in almost all statistical computer packages, age-specific reference intervals may be obtained. The scope of the method is illustrated by fitting models to several real data sets and assessing each model using goodness-of-fit techniques.

Adolescent↗

PubMed1997

Thyroid Na+/I- symporter. Mechanism, stoichiometry, and specificity.

The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ >> Li+ >> H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM and 33 +/- 9 microM, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (ClO4-) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (approximately 35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8-14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Boltzmann relation. The voltage for half-maximal charge translocation (V0.5) was -15 +/- 3 mV, and the apparent valence of the movable charge was 1. Total charge was insensitive to [Na+]o, but V0.5 shifted to more negative potentials as [Na+]o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was >/=22 s-1 in the Na+ uniport mode and >/=36 s-1 in the Na+/I- cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a approximately 2. 5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first.

Animals↗

PubMed1997

Membrane topology motifs in the SGLT cotransporter family.

Homologues of the Na+/glucose cotransporter, the SGLT family, include sequences of mammalian, eubacterial, yeast, insect and nematode origin. The cotransported substrates are sugars, inositol, proline, pantothenate, iodide, urea and undetermined solutes. It is reasonable to expect that the SGLT family members share a similar or identical topology of membrane spanning elements, by virtue of their common ancestry and similar coupling of solute transport to downhill sodium flux. Here we examine their membrane topologies as deduced from diverse analyses of their primary sequences, and from their sequence correlations with the experimentally determined topology of the human Na+/ glucose contransporter SGLT1. Our analyses indicate that all family members share a common core of 13 transmembrane helices, but that some, like SGLT1 itself, have one additional span appended to the C-terminus, and still others, two. One bacterial member incorporates an additional span at the N-terminus. Sequence comparisons indicative of common ancestry of the SGLT and the [Na+ + Cl-] transporter families are introduced, and evaluated in light of their topologies. New evidence concerning the previously asserted common ancestry of SGLT1 and an N-acetylglucosamine permease of the bacterial phosphotransferase system is considered. Finally, we analyze observations which lead us to conjecture that the experimental strategy most commonly employed to reveal the topology of bacterial transporters (i.e., the fusion of reporter enzymes such as phoA alkaline phosphatase, beta-lactamase or beta-galactosidase, to progressively C-truncated fragments of the transporter) has often instead so perturbed local topology as to have entirely missed pairs of adjacent membrane spans.

Amino Acid Sequence↗

PubMed1997

Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter.

To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C5-cRNA injection compared with noninjected oocytes. The diameters of the C5 particles were heterogeneous (4.8 +/- 0.3, 7.1 +/- 1.2, and 10.3 +/- 0.8 nm) in contrast to the endogenous particles (7.6 +/- 1.2 nm). C5 increased the alpha-methyl-D-glucopyranoside (alphaMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5alphaMDG of 50 mM and a turnover of approximately 660 s-1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+: 1) selective (alphaMDG > D-glucose > D-galactose >> L-glucose approximately D-mannose), 2) inhibited by phloretin, KiPT = approximately 500 microM, and 3) insensitive to phlorizin. These results indicate that C5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC5 (the human equivalent of C5), hC4 (human SGLT1 amino acids 407-648, helices 10-13), and hN13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.

Animals↗

PubMed1997

Cation effects on protein conformation and transport in the Na+/glucose cotransporter.

Cation-driven cotransporters are essential membrane proteins in procaryotes and eucaryotes, which use the energy of the transmembrane electrochemical gradient to drive transport of a substrate against its concentration gradient. Do they share a common mechanism? Cation selectivity of the rabbit isoform of the Na+/glucose cotransporter (SGLT1) was examined using the twoelectrode voltage clamp and the Xenopus oocyte expression system. The effect of H+, Li+, and Na+ on kinetics of SGLT1 was compared to the effects of these cations on the bacterial melibiose. In SGLT1, substitution of H+ or Li+ for Na+ caused a kinetic penalty in that the apparent affinity for sugar (K0.5sugar) decreased by an order of magnitude or more (from 0.2 to 30 mM) depending on the membrane potential and cation. The effect of the cation on the K0.5sugar/V profiles was independent of the sugar for glucose and alpha-methyl-beta-D-glucose; this profile was maintained for galactose in Li+ and Na+, but was 2 orders of magnitude higher in H+, but the Imax for glucose, galactose, and alpha-methyl-beta-D-glucose in a given cation were identical. Li+ supported a lower maximal rate of transport (Imax) than Na+ (approximately 80% of ImaxNa), while the Imax in H+ was higher than Na+ (>/=180% of ImaxNa). Our interpretation of these results and simulations using a six-state mathematical model, are as follows. 1) Binding of the cation causes a conformational change in the sugar binding pocket, the exact conformation being determined by the specific cation. 2) Once the sugar is bound, it is transported at a characteristic rate determined by the cation. 3) Mathematical simulations suggest that the largest contribution to the kinetic variability of both cation and sugar transport is associated with cation binding. Similarity to the effects of cation substitution in MelB suggests that the mechanism of energy coupling has been evolutionarily conserved.

Animals↗

PubMed1997

Presteady-state currents of the rabbit Na+/glucose cotransporter (SGLT1).

The rabbit Na+/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na+, membrane voltage Vm, phlorizin and temperature. Step changes in membrane voltage-from the holding Vh to test values, elicited transient currents that rose rapidly to a peak (at 3-4 msec), before decaying to the steady state, with time constants tau approximately 4-20 msec, and were blocked by phlorizin (Ki approximately 30 microm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of Vm. The Q-V curves obeyed the Boltzmann relation: the maximal charge Qmax was 4-120 nC; V0.5, the voltage for 50% Qmax was -5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Qmax and z were independent of Vh (between 0 and -100 mV) and temperature (20-30 degrees C), while increasing temperature shifted V0.5 towards more negative values. Decreasing [Na+]o decreased Qmax, and shifted V0.5 to more negative voltages 9by -100 mV per 10-fold decrease in [Na+]o). The time constant tau was voltage dependent: the tau-V relations were bell-shaped, with maximal taumax 8-20 msec. Decreasing [Na+]o decreased taumax, and shifted the tau-V curves towards more negative voltages. Increasing temperature also shifted the tau-V curves, but did not affect taumax. The maximum temperature coefficient Q10 for tau was 3-4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na+/glucose cotransport indicate that charge-movements are due to Na+-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the tau-V relations are bell-shaped, and shift towards more negative voltages as [Na+]o is reduced; (iii) taumax is decreased with decreasing [Na+]o; and (iv) the Q-V relations are shifted towards negative voltages as [Na+]o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model.

Animals↗

PubMed1997

Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects.

BACKGROUND & AIMS: Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose-galactose malabsorption, characterized by severe diarrhea. This study focused on a unique proband with glucose-galactose malabsorption who was investigated 30 years ago, and the aims of the study were to identify mutations in the SGLT1 gene and to determine the defect in sugar transport. METHODS: Mutations were identified by sequencing, and each mutant protein was then studied using a Xenopus oocyte heterologous expression system. Analysis included Western, freeze fracture, radiotracer uptake, and electrophysiological assays. RESULTS: Two heterozygous missense mutations (Cys355Ser and Leu147Arg) were identified that entirely eliminated Na+/sugar cotransport activity. Western blot analysis showed that the levels of both mutant proteins in the oocyte were comparable to wild-type SGLT1, but no complex glycosylation was detected. No SGLT1 charge movements were observed with the mutant proteins, and freeze fracture data showed that neither mutant protein reached the plasma membrane. CONCLUSIONS: The Cys355Ser and Leu147Arg mutations eliminate the Na+/sugar cotransport by blocking the transfer of SGLT1 protein from the endoplasmic reticulum to the plasma membrane. This is consistent with earlier studies on phlorizin binding to the brush border membrane of duodenal biopsy specimens from this patient.

Amino Acid Sequence↗