Biomedical subjects

E M Wright

Publications and source records attributed to E M Wright.

At least 55 records · Page 3Linked to original sources

PubMed2000

Neutralization of a conserved amino acid residue in the human Na+/glucose transporter (hSGLT1) generates a glucose-gated H+ channel.

The role of conserved Asp204 in the human high affinity Na+/glucose cotransporter (hSGLT1) was investigated by site-directed mutagenesis combined with functional assays exploiting the Xenopus oocyte expression system. Substitution of H+ for Na+ reduces the apparent affinity of hSGLT1 for glucose from 0.3 to 6 mm. The apparent affinity for H+ (7 microm) is about three orders of magnitude higher than for Na+ (6 mm). Cation/glucose cotransport exhibits a coupling ratio of 2 Na+ (or 2 H+):1. Pre-steady-state kinetics indicate that similar Na+ - or H+ -induced conformational changes are the basis for coupled transport. Replacing Asp204 with Glu increases the apparent affinity for H+ by >20-fold with little impact on the apparent Na+ affinity. This implies that the length of the carboxylate side chain is critical for cation selectivity. Neutralization of Asp204 (Asp --> Asn or Cys) reveals glucose-evoked H(+) currents that were one order of magnitude greater than Na(+) currents. These phlorizin-sensitive H+ currents reverse and are enhanced by internal acidification of oocytes. Together with a H(+) to sugar stoichiometry as high as 145:1, these results favor a glucose-gated H+ channel activity of the mutant. Our observations support the idea that cotransporters and channels share common features.

Base Sequence↗

PubMed2000

Characterization of the Vibrio parahaemolyticus Na+/Glucose cotransporter. A bacterial member of the sodium/glucose transporter (SGLT) family.

The Vibrio parahaemolyticus sodium/glucose transporter (vSGLT) is a bacterial member of the SGLT gene family. Wild-type and mutant vSGLT proteins were expressed in Escherichia coli, and transport activity was measured in intact cells and plasma membrane vesicles. Two cysteine-less vSGLT proteins exhibited sugar transport rates comparable with that of the wild-type protein. Six residues in two regions of vSGLT known to be of functional importance in SGLT1 were replaced individually with cysteine in the cysteine-less protein. Characterization of these single cysteine-substituted vSGLTs showed that two residues (Gly-151 and Gln-428) are essential for transport function, whereas the other four residues (Leu-147, Leu-149, Ala-423, and Gln-425) are not. 2-Aminoethylmethanethiosulfonate (MTSEA) blocked Na(+)/glucose transport by only the transporter bearing a cysteine at position 425 (Q425C). MTSEA inhibition was reversed by dithiothreitol and blocked by the presence of both Na(+) and d-glucose, indicating that conformational changes of the vSGLT protein are involved in Na(+)/glucose transport. A split version of vSGLT was generated by co-expression of the N-terminal (N(7)) and C-terminal (C(7)) halves of the transporter. The split vSGLT maintained Na(+)-dependent glucose transport activity. Chemical cross-linking of split vSGLT, with a cysteine in each N(7) and C(7) fragment, suggested that hydrophilic loops between helices 4 and 5 and between helices 10 and 11 are within 8 A of each other. We conclude that the mechanism of Na(+)/glucose transport by vSGLT is similar to mammalian SGLTs and that further studies on vSGLT will provide novel insight to the structure and function of this class of cotransporters.

Bacterial Proteins↗

PubMed2000

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.

The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.

Base Sequence↗

PubMed2000

Pentameric assembly of a neuronal glutamate transporter.

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure models (6-10 transmembrane alpha-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110 degrees. Each domain appeared to contribute to an extracellular mass that projects approximately 3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.

Amino Acid Transport System X-AG↗

PubMed2000

Glycoside binding and translocation in Na(+)-dependent glucose cotransporters: comparison of SGLT1 and SGLT3.

Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na(+)/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K(0.5) 0.06 mm) and 2-naphthylglucose with lower affinity (K(0.5) 0. 5 mm) than alpha-methyl-d-glucopyranoside (alpha MDG, 0.2 mm). Both were poorly transported (maximal velocities, I(max), 14% and 8% of alpha MDG). Other compounds were inhibitors (K(i)s 1-13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than alpha MDG (K(0.5)s 0.9, 0.2 and 2.5 mm and relative I(max)s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I(max)s (130 and 120%) and comparable K(0. 5)s (8 and 1 mm). Increased affinity of indican relative to alphaMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value.

Animals↗

PubMed2000

Dark solitons in a one-dimensional condensate of hard core bosons.

A mapping theorem leading to exact many-body dynamics of impenetrable bosons in one dimension reveals dark and gray solitonlike structures in a toroidal trap which is phase imprinted. On long time scales revivals appear that are beyond the usual mean-field theory.

Journal Article↗

PubMed2000

Proteomics on full-length membrane proteins using mass spectrometry.

A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.

Alkylation↗

PubMed2000

Graphical exploration of covariate effects on survival data through nonparametric quantile curves.

Kaplan-Meier curves provide an effective means of presenting the distributional pattern in a sample of survival data. However, in order to assess the effect of a covariate, a standard scatterplot is often difficult to interpret because of the presence of censored observations. Several authors have proposed a running median as an effective way of indicating the effect of a covariate. This article proposes a form of kernel estimation, employing double smoothing, that can be applied in a simple and efficient manner to construct an estimator of a percentile of the survival distribution as a function of one or two covariates. Permutations and bootstrap samples can be used to construct reference bands that help identify whether particular features of the estimates indicate real features of the underlying curve or whether this may be due simply to random variation. The techniques are illustrated on data from a study of kidney transplant patients.

Biometry↗

PubMed2000

Coupling between Na+, sugar, and water transport across the intestine.

Water is absorbed across the small intestine in the absence of external driving forces. However, it has been established that water transport is secondary to active sodium transport. In the upper intestine both sodium and water absorption are largely dependent on the presence of D-glucose. The link between active sodium transport and glucose is the coupled transport of sodium and glucose across the brush border membrane of enterocytes by the Na+/glucose cotransporter (SGLT1). Na+ that enters the cells with glucose is pumped out towards the blood by 3Na+/2K+ pumps on the basolateral membrane, and glucose passes out across the basolateral membrane by facilitated diffusion, the net result being that glucose and sodium are transported across the epithelium. The coupling between Na+, glucose, and water transport is less well understood. It is commonly thought that Na+ transport increases the local osmotic pressure in the lateral intercellular spaces, and that this in turn generates osmotic water flow across the epithelium. Recent work suggests a more direct link between Na+, glucose, and water transport; that is, water is cotransported along with Na+ and sugar through SGLT1. Here we review the evidence for Na+/glucose/water cotransport.

Animals↗

PubMed2000

Regulation of the human Na(+)-glucose cotransporter gene, SGLT1, by HNF-1 and Sp1.

The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.

Animals↗

PubMed2000

Water transport by the renal Na(+)-dicarboxylate cotransporter.

This study investigated the ability of the renal Na(+)-dicarboxylate cotransporter, NaDC-1, to transport water. Rabbit NaDC-1 was expressed in Xenopus laevis oocytes, cotransporter activity was measured as the inward current generated by substrate (citrate or succinate), and water transport was monitored by the changes in oocyte volume. In the absence of substrates, oocytes expressing NaDC-1 showed an increase in osmotic water permeability, which was directly correlated with the expression level of NaDC-1. When NaDC-1 was transporting substrates, there was a concomitant increase in oocyte volume. This solute-coupled influx of water took place in the absence of, and even against, osmotic gradients. There was a strict stoichiometric relationship between Na(+), substrate, and water transport of 3 Na(+), 1 dicarboxylate, and 176 water molecules/transport cycle. These results indicate that the renal Na(+)-dicarboxylate cotransporter mediates water transport and, under physiological conditions, may contribute to fluid reabsorption across the proximal tubule.

Animals↗

PubMed1999

Purification and functional reconstitution of a truncated human Na(+)/glucose cotransporter (SGLT1) expressed in E. coli.

A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture. Reconstitution of both GST-C(5) and C(5) proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [(3)H]D-glucose into C(5)-proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C(5) in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.

Escherichia coli↗

PubMed1999

Number of subunits comprising the epithelial sodium channel.

The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, alpha, beta, and gamma. Understanding the structure and function of this channel and its abnormal behavior in disease requires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological methods to evaluate the number of subunits in the ENaC complex expressed in Xenopus laevis oocytes. In oocytes expressing wild-type hENaC (alpha, beta, and gamma subunits), clusters of particles appeared in the protoplasmic face of the plasma membrane. The total number of particles in the clusters was consistent with the whole-cell amiloride-sensitive current measured in the same cells. The size frequency histogram for the particles in the clusters suggested the presence of an integral membrane protein complex composed of 17 +/- 2 transmembrane alpha-helices. Because each ENaC subunit has two putative transmembrane helices, these data suggest that in the oocyte plasma membrane, the ENaC complex is composed of eight or nine subunits. At high magnification, individual ENaC particles exhibited a near-square geometry. Functional studies using wild-type alphabeta-hENaC coexpressed with gamma-hENaC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and cadmium, suggested that the functional channel complex contains more than one gamma subunit. These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are gamma subunits.

Animals↗

PubMed1999

Analysing repeated measurements data: a practical comparison of methods.

A variety of methods are available for analysing repeated measurements data where the outcome is continuous. However, there is little information on how established methods, such as summary statistics and repeated measures analysis of variance (RMAOV), compare in practice with methods that have become available to applied statisticians more recently, such as marginal models (based on generalized estimating equation methodology) and multilevel models (that is, hierarchical random effects models). The aim of this paper is to exemplify the use of these methods, and directly compare their results by application to a clinical trial data set. The focus is on practical aspects rather than technical issues. The data considered were taken from a clinical trial of treatments for asthma in 240 children, in which a baseline and four post-randomization measurements of outcomes were taken. The simplicity of the method of summary statistics using the post-randomization mean of observations provided a useful initial analysis. However, fixed time effects or treatment-time interactions cannot be included in such an analysis, and choice of appropriate weighting when there is substantial missing data is problematic. RMAOV, marginal models and multilevel models generally provided similar estimates and standard errors for the treatment effects, although in one example with a relatively complex variance structure the marginal model produced less efficient estimates. Two advantages of multilevel models are that they provide direct estimates of variance components which are often of interest in their own right, and that they can be naturally extended to handle multivariate outcomes.

Adolescent↗

PubMed1999

Passive water and ion transport by cotransporters.

1. The rabbit Na+-glucose (SGLT1) and the human Na+-Cl--GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker-sensitive Na+ currents in the absence of substrates (glucose and GABA). 2. Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3-5 microM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 microM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 3. The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol-1, respectively. The high Ea for Na+ transport is comparable to that of Na+-glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 4. GAT1 also behaved as an SKF89976A-sensitive water channel. We did not observe passive Na+ transport through GAT1. 5. We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.

Animals↗

PubMed1999

Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects.

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.

Cell Membrane↗

PubMed1999

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes.

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.

Animals↗

PubMed1999

Fetal ultrasound biometry: 1. Head reference values.

OBJECTIVE: To create reliable reference ranges and calculate Z scores for fetal head 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 exactly 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 6217 fetal head biparietal diameters and 5510 occipito-frontal diameters were measured. Both head circumference and cephalic index were derived in 5462 cases where both biparietal diameter and occipito-frontal diameter could be measured on the same fetus. The centile charts, tables and regression formulae for biparietal and occipito-frontal diameters, head circumference and cephalic index are presented. An application to calculate Z scores was developed using Excel (Microsoft Corporation, USA) and macros are presented in detail in the Figure 8 footnote. The comparison of our charts with those of the two most recent studies revealed almost no differences in biparietal diameter centiles. In one publication, occipito-frontal diameter charts, and in another, head circumference charts were different from the current study. CONCLUSIONS: We have presented centile charts, tables and regression formulae for fetal head ultrasound biometry 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 for use in computer-aided evaluation of fetal ultrasound biometry.

Adult↗