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E M Wright

Publications and source records attributed to E M Wright.

At least 145 records · Page 8Linked to original sources
PubMed1992

Molecular biology approaches to comparative study of Na(+)-glucose cotransport.

The rabbit intestinal Na(+)-glucose cotransporter has been cloned and sequenced. The cDNA encoding the cotransporter has been used in two general lines of research related to comparative aspects of Na(+)-glucose cotransport that are reviewed here. First, defined regions of the predicted amino acid sequence were used to raise antibodies, and the species distribution of epitopes recognized by those antibodies was investigated. Intestinal brush-border membranes from mammals, birds, an amphibian, and a reptile were all found to contain protein that were recognized by the antibodies in Western analysis. The cDNA encoding the rabbit intestinal Na(+)-glucose cotransporter was also used directly to examine the species distribution of related mRNA in Northern studies and to isolate new cDNAs encoding other Na(+)-glucose cotransporters. Northern blots revealed the presence of related mRNAs in intestines of mammals and a fish, as well as rabbit kidney and gills of marine mussels. The cDNAs encoding mammalian Na(+)-glucose cotransporters and bacterial Na(+)-dependent cotransporters for proline and pantothenate share sequence homology. There has been evolutionary conservation of the structure and function of the Na(+)-glucose cotransporter, and there appears to be a gene family that codes for the Na(+)-coupled cotransporters.

Amino Acid Sequence↗
PubMed1992

Cloning of a human kidney cDNA with similarity to the sodium-glucose cotransporter.

We have used low-stringency screening with the human intestinal Na(+)-glucose cotransporter SGLT1 to isolate a 2,271-nucleotide cDNA (Hu14) from human kidney. This clone, which encodes a 672-residue protein, is 59% identical at the amino acid level to SGLT1 and has a similar number and arrangement of predicted membrane-spanning regions. It also shares significant sequence identity with other Na(+)-coupled transporters. Northern blot analysis suggests strong expression of Hu14 in kidney, but, unlike SGLT1, no significant expression in intestine. We have been unable to demonstrate definitive transport of any of a number of substrates (including amino acids, sugars, nucleosides, and vitamins) into Hu14 cRNA-injected Xenopus oocytes, although sequence conservation makes it likely that Hu14 represents another member of the Na+ cotransporter family, possibly a second Na(+)-glucose cotransporter.

Amino Acid Sequence↗
PubMed1992

Continuous low-dose 3-in-1 nerve blockade for postoperative pain relief after total knee replacement.

We have investigated the value of a 3-in-1 nerve block, followed by a continuous low-dose infusion of bupivacaine into the femoral nerve sheath for postoperative analgesia after total knee replacement. Thirty-seven patients were randomly allocated to either a control group or a study group. The study group had a catheter placed in the ipsilateral femoral nerve sheath. A 3-in-1 nerve block was then performed in the study group with injection of 30 mL of 0.25% bupivacaine through the catheter. This was followed by a continuous infusion of 0.125% bupivacaine at 6 mL/h. The study group had significantly lower pain scores 4 and 24 h postoperatively (P less than 0.01) and required less postoperative opioid analgesic medication (P less than 0.01) than the control group. The authors conclude that a continuous low-dose infusion into the femoral nerve sheath results in better pain relief than conventional intramuscularly administered narcotics after total knee arthroplasty.

Aged↗
PubMed1992

Colocalization of GLUT2 glucose transporter, sodium/glucose cotransporter, and gamma-glutamyl transpeptidase in rat kidney with double-peroxidase immunocytochemistry.

Glucose is reabsorbed from the glomerular filtrate in the proximal segment of the renal tubule in two stages. The first stage is uphill transport across the brush border membrane by Na(+)-glucose cotransport and the second stage is downhill transport across the basolateral membrane by facilitated diffusion. Genes for both a renal Na(+)-glucose cotransporter (SGLT1) and a renal facilitated glucose transporter (GLUT2) have been cloned and sequenced. To examine whether SGLT1 and GLUT2 colocalize to the same tubular epithelial cells in rat kidney, double-immunoperoxidase studies with dual chromogens and paraformaldehyde perfusion-fixed frozen sections of rat kidney were performed. Antipeptide antisera were prepared against rat GLUT2 (amino acids 510-522) and rabbit SGLT1 (amino acids 402-420). Proximal tubules were identified immunocytochemically with an antiserum raised against a synthetic peptide corresponding to the 21 amino acids at the COOH-terminal of the heavy chain of rat gamma-glutamyl transpeptidase, which is a proximal tubule-specific enzyme. The anti-GLUT2 antiserum strongly stained the basolateral membrane of 46% of cortical tubules, whereas the SGLT1 antiserum stained the brush border of 56% of the cortical tubules. The gamma-glutamyl transpeptidase antiserum also stained the brush border of 51% of the cortical tubules. GLUT2 and SGLT1 colocalized to 40% of cortical epithelium, but 16% of cortical epithelial cells were immunopositive for brush border SGLT1 and immunonegative for basolateral GLUT2. These gamma-glutamyl transpeptidase staining results suggest that at least 50% of the tubules in the cortex are proximal tubules and that SGLT1 and GLUT2 colocalize to most proximal tubules. The fact that SGLT1 antiserum immunoreacted with tubules unreactive to the GLUT2 antiserum suggests that either the SGLT1 epitope is conserved on a related brush border protein or that there is another GLUT transporter responsible for the exit of sugar from these proximal tubule cells.

Animals↗
PubMed1992

The Na+/glucose cotransporter (SGLT1).

An important class of Na+ transport proteins is the cotransporters. They exist in bacteria and animal cells and are responsible for the "active" accumulation of sugars, amino acids, carboxylic acids and some ions, e.g., I-, Cl-, and PO-4, in cells. In the small intestine and renal proximal tubule the cotransporters play an important role in the transport of salt and water across the epithelia. The most well known and best characterized Na+ cotransporter is the intestinal brush border Na+/glucose cotransporter. We have cloned, sequenced, and expressed both the rabbit and human Na+/glucose cotransporters. The cDNAs code for 73kDa proteins with 662-664 residues (86% identity). Secondary structure analysis suggests a 12 membrane-spanning helical model with the N- and C-termini in the cytoplasm. A single N-linked glycosylation site is utilized at Asn248. These sugars are not required for function. Two essential residues for functional expression in oocytes have been identified, Asp28 and Arg300. In two sisters with glucose-galactose malabsorption the transport defect is caused by a missense mutation changing Asp28 to Asn28, and we have found that changing Arg300 to Cys300 eliminated transport. Current research is directed to finding residues and domains essential for ligand binding and transport, and we are using electrophysiological techniques to correlate structure and function.

Amino Acid Sequence↗
PubMed1992

Milrinone in the treatment of low output states following cardiac surgery.

Milrinone is known to have beneficial haemodynamic and clinical effects in patients with congestive heart failure. An investigation into the safety and efficacy of milrinone in patients following heart surgery was undertaken by the European Milrinone Multicentre Trial Group. This paper reports the efficacy, the effects on left heart function, and the adverse events in the study. Ninety-nine adult patients, 61 coronary artery bypass grafting (CABG), 33 valve surgery (VS), and five CABG+VS were studied. Three dosage regimens were investigated sequentially. All patients received a loading dose of intravenous milrinone 50 micrograms kg-1 over 10 min, followed by an infusion of either 0.375 micrograms kg-1 min-1, 0.5 microgram kg-1 min-1, or 0.75 microgram kg-1 min-1 over 12 h. The groups were comparable for age, weight, and surface area; however, in the group receiving 0.5 microgram kg-1 min-1 there were more females and patients undergoing mitral valve surgery. Efficacy criteria of an increase in cardiac index of 30% and/or a decrease in mean pulmonary capillary wedge pressure of 25% were fulfilled by 77 patients at the 60-min measurement. Of the remaining 22 patients, 17 were clinically satisfactory and fulfilled efficacy criteria at some time during the study. At 15 min and 60 min there was a dose-related decrease in systolic and diastolic arterial pressure; however, there was no significant difference in the mean arterial pressure measurements. In all groups there was an improvement in cardiac index at 15 min following the start of milrinone, which was sustained during and up to 4 h after the infusion. This was closely associated with changes in stroke volume index and systemic vascular resistance, and not solely due to a change in heart rate.

Adult↗
PubMed1991

Distribution of the SGLT1 Na+/glucose cotransporter and mRNA along the crypt-villus axis of rabbit small intestine.

The expression of the Na+/glucose cotransporter (SGLT1) mRNA and protein along the crypt-villus axis of the rabbit small intestine was examined using in situ hybridization and immunocytochemical techniques. We detected mRNA in the cells on the villus, but not in the crypts, and the mRNA abundance increased 6-fold from the base to the tip of the villus. SGLT1 protein was restricted to the brush borders of mature enterocytes. We suggest that the high rate of sugar transport across the tips of the villus is due to the transcription of the SGLT1 gene in mature enterocytes, the subsequent translation of SGLT mRNA, and the insertion direct of the functional SGLT1 transporter into the brush border membrane of these cells lining the villus tip.

Animals↗
PubMed1991

Biosynthesis of the cloned intestinal Na+/glucose cotransporter.

The initial stages in the biosynthesis of the cloned Na+/glucose cotransporter were examined by in vitro expression of the protein in the absence and presence of pancreatic microsomes. Glycosylation was detected by endoglycosidase-H shifts in the apparent size of the proteins on SDS-PAGE. In the presence of microsomes, Mr increased from 52,000 to 58,000, and this was reversed by endo-H. This demonstrates that the protein is glycosylated and that there is no large cleavable signal sequence. Using partial transcripts and site-directed mutagenesis, we established that Asn-248 is glycosylated. Glycosylation was not required for the functional expression of the transporter in Xenopus oocytes. In terms of the topology of the protein, these results suggest that Asn-248 is on the external surface of the membrane.

Acetylglucosaminidase↗
PubMed1991

Glucose/galactose malabsorption caused by a defect in the Na+/glucose cotransporter.

Glucose/galactose malabsorption (GGM) is an autosomal recessive disease manifesting within the first weeks of life and characterized by a selective failure to absorb dietary glucose and galactose from the intestine. The consequent severe diarrhoea and dehydration are usually fatal unless these sugars are eliminated from the diet. Intestinal biopsies of GGM patients have revealed a specific defect in Na(+)-dependent absorption of glucose in the brush border. Normal glucose absorption is mediated by the Na+/glucose cotransporter in the brush border membrane of the intestinal epithelium. Cellular influx is driven by the transmembrane Na+ electrochemical potential gradient; thereafter the sugar moves to the blood across the basolateral membrane via the facilitated glucose carrier. We have previously cloned and sequenced a Na+/glucose cotransporter from normal human ileum and shown that this gene, SGLT1, resides on the distal q arm of chromosome 22. We have now amplified SGLT1 complementary DNA and genomic DNA from members of a family affected with GGM by the polymerase chain reaction. Sequence analysis of the amplified products has revealed a single missense mutation in SGLT1 which cosegregates with the GGM phenotype and results in a complete loss of Na(+)-dependent glucose transport in Xenopus oocytes injected with this complementary RNA.

Antisense Elements (Genetics)↗
PubMed1991

Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine.

Inward Na+ currents associated with the cloned intestinal Na+/glucose cotransporter expressed in Xenopus oocytes have been studied using the two-microelectrode voltage-clamp method. The steady-state current/voltage relations showed voltage-dependent (Vm from +20 to -75 mV) and relatively voltage-independent (Vm from -75 to -150 mV) regions. The apparent Imax for Na+ and glucose increased with negative membrane potentials, and the apparent K0.5 for glucose (K(Glc)0.5) depended on Vm and [Na]o. Increasing [Na]o from 7 to 110 mmol/l had the same effect in decreasing K(Glc)0.5 from 0.44 to 0.03 mmol/l as increasing the Vm from -40 to -150 mV. The I/V curves under saturating conditions (20 mmol/l external sugars and 110 mmol/l [Na]o) were identical for D-glucose, D-galactose, alpha-methyl D-glucopyranoside and 3-O-methyl D-glucoside. The specificity of the cotransporter for sugars was: D-glucose, D-galactose, alpha-methyl D-glucopyranoside greater than 3-O-methyl D-glucoside much greater than D-xylose greater than D-allose much greater than D-mannose. Ki for phlorizin (approximately 10 mumol/l) was independent of Vm at saturating [Na]o. We conclude that a variety of sugars are transported by the cloned Na+/glucose cotransporter at the same maximal rate and that membrane potential affects both the maximal current and the apparent K0.5 of the cotransporter for Na+ and glucose.

Animals↗
PubMed1991

Clinical and haemodynamic effects of milrinone in the treatment of low cardiac output after cardiac surgery.

We have studied the haemodynamic effects of i.v. milrinone, a new phosphodiesterase inhibitor, in patients with low cardiac output after cardiac surgery. Thirty-five patients with a cardiac index (Cl) less than 2.5 litre min-1 m-2 and a pulmonary capillary wedge pressure (PCWP) greater than 8 mm Hg were given a loading dose of milrinone 50 micrograms kg-1 followed by an infusion at one of three rates: 0.375 micrograms kg-1 min-1, 0.5 micrograms kg-1 min-1 or 0.75 micrograms kg-1 min-1 for 12 h. After 1 h there were increases in Cl (35%) (P less than 0.001), heart rate (13%) (P less than 0.01) and stroke volume index (19%) (P less than 0.005). There were decreases in mean arterial pressure (12%) (P less than 0.01), systemic vascular resistance (35%) (P less than 0.001) and PCWP (24%) (P less than 0.05). Pulmonary vascular resistance was unchanged or reduced and left ventricular stroke work index was unchanged or increased. The haemodynamic improvements were sustained throughout the infusion period. Milrinone was tolerated well: three patients developed tachycardia greater than 125 beat min-1, one patient developed atrial fibrillation and one patient had a short run of atrial bigemini. We conclude that milrinone is a useful agent in the treatment of patients with a reduced cardiac output after cardiac surgery.

Adult↗
PubMed1991

Ontogenic development of lamb intestinal sodium-glucose co-transporter is regulated by diet.

1. The ontogenic development of the intestinal Na(+)-glucose co-transporter was measured in lambs as a function of diet. Transport activity was assayed in brush-border membrane vesicles and the expression of transport protein in the brush-border membrane determined by Western analysis. 2. Na(+)-dependent D-glucose transport increased to a maximum (300-700 pmol mg-1 s-1) within the first 2 weeks of birth and then declined to negligible amounts (less than 10 pmol mg-1 s-1) over the next 8 weeks. There was no further change over the next 2-3 years. Early changes were associated with modifications in both the maximum velocity Vmax for transport and expression of carrier protein in the brush-border plasma membrane. 3. Maintaining lambs on a milk replacer diet beyond the normal weaning period prevented the normal decline in the expression of Na(+)-glucose co-transport. At 5 weeks the transport rate was 433 +/- 150 pmol mg-1 s-1 in lambs maintained on milk replacer, but only 79 +/- 40 pmol mg-1 s-1 in normally reared control lambs. 4. Infusing the proximal intestine of 2- to 3-year-old sheep with 30 mM-D-glucose for four days increased the rate of transport 40- to 80-fold above that found in control animals perfused with mannitol. A similar but smaller increase was observed in one animal perfused with the non-metabolizable sugar alpha-methyl-D-glucopyranoside. The induced increase in glucose transport was correlated with the expression of the co-transporter protein in the brush-border plasma membrane. 5. It is concluded that the age-related decline in Na(+)-glucose co-transport in the sheep intestine is directly due to the decrease in D-glucose (and D-galactose) reaching the small intestine after development of the rumen. These results further suggest that luminal sugar substrates for the co-transporter promote both the maintenance and the up-regulation of the brush-border transport protein and it is the intact sugar itself which controls gene expression during enterocyte maturation.

Animals↗
PubMed1991

Mitral valve repair: a valuable procedure with good long term results even when performed infrequently.

OBJECTIVE: To assess the results of mitral valve repair in a series with a low frequency of repair despite a policy to conserve the valve whenever possible. DESIGN: Retrospective review of case notes with clinical and echocardiographic examination of survivors. SETTING: Cardiac surgery unit in a university teaching hospital. PATIENTS: 62 consecutive patients undergoing mitral valve repair by one surgeon between 1979 and 1989. INTERVENTIONS: Mitral valve repair according to the criteria and techniques of Carpentier. MAIN OUTCOME MEASURES: Frequency of repair, operative mortality, actuarial survival, freedom from reoperation and thromboembolism, clinical state, and echocardiographic state. RESULTS: The 62 patients, median age 58 years (interquartile range 51 to 64 years), represent 14% (70% confidence interval 12% to 15%) of the 454 mitral valve operations performed in the study period. Operative mortality was 8% (70% CI 5% to 13%). Actuarial survival was 62% (70% CI 43% to 81%) at nine years. At nine years actuarial freedom from reoperation was 91% (70% CI 79% to 102%) and freedom from thromboembolism 91% (70% CI 80% to 102%). At a median follow up of 33 months (interquartile range 21 to 74 months), 38 of 46 survivors had improved functional state. Of 21 patients who underwent echocardiography one had severe mitral regurgitation and one mitral stenosis. CONCLUSION: Although compared with other reports of mitral valve repair the prevalence of repair was low in this series the results are comparable and justify a positive approach to repair in all patients undergoing mitral valve surgery even if this can only be achieved in a small proportion of patients.

Female↗
PubMed1991

Intestinal and renal Na+/glucose cotransporters share common structures.

Polyclonal antibodies were raised to peptides selected from three different regions of the cloned rabbit intestinal Na+/glucose cotransporter. Western blot analysis was used to identify the fully mature protein in intestinal and renal brush borders. Two of the antibodies specifically identified a approximately 70-kDa protein band in rabbit intestinal brush borders but did not specifically immunoreact with membranes that do not have Na(+)-dependent glucose transport activity. The immunoreactive proteins had an apparent isoelectric point between pH 4.7 and 6.8. The antibodies also specifically recognized a similar-sized protein in human and seven other mammalian intestinal brush borders. Similar protein bands were identified in four mammalian renal brush-border membranes, indicating shared epitopes between intestinal and renal cotransport proteins. In some species, e.g., lamb and pig, the epitope for one antibody was missing in both intestinal and renal brush borders, suggesting that this epitope is not essential for function. These results suggest that 1) the cloned intestinal Na+/glucose cotransporter is that identified in earlier biochemical studies, 2) there is close structural similarity between intestinal and renal cotransporters, and 3) the structure of these proteins has been conserved during evolution.

Animals↗
PubMed1990

Expression of mammalian renal transporters in Xenopus laevis oocytes.

We have injected mRNA from rabbit renal cortex into Xenopus oocytes and demonstrated the expression of renal carriers for Na(+)-independent transport of L-phenylalanine and L-lysine and Na(+)-dependent transport of L-alanine and succinate. Maximal expression of renal amino acid transporters occurred 6-8 days following mRNA injection. The proteins responsible for transport of these four substrates were translated from mRNAs which are between 1.5 and 3.0 kb. This information serves as a starting point for expression cloning of these transport proteins.

Amino Acid Transport Systems↗