A simple liposomal system to reconstitute and assay highly efficient Na⁺/D-glucose cotransport from kidney brush-border membranes

A simple procedure to reconstitute highly efficient Na+/D-glucose cotransport from solubilized brush-border membranes of proximal kidney tubules is described. Reconstitution of transport activity was possible with various phospholipid and cholesterol combinations; the presence, however, of cholesterol and at least one phospholipid was essential. When liposomes were synthesized from only one phospholipid and cholesterol, the highest uptake rats were observed with phosphatidylserine; phosphatidylcholine was less effective and phosphatidylethanolamine showed insignificant uptake of D-glucose in the presence of Na+. The rate at which an inward-directed Na+ gradient dissipated across the liposomal membranes was reduced if the cholesterol concentration of liposomes was increased. In the optimized system, proteoliposomes were formed from cholesterol and phosphatidylserine by a heat-sonication-freeze-thaw procedure. A Na+-gradient persisted for hours across these proteoliposomal membranes and a Na+/D-glucose cotransport with the following characteristics could be demonstrated: (1) dependency on the Na+ gradient; (2) a transient (3) rheogenicity; (4) stereospecificity; and (5) high-affinity phlorizin inhibition. Since the Na+-gradient-stimulated D-glucose uptake is linear for minutes, the initial uptake rates can be measured and the Na+/D-glucose cotransport activity of different protein fractions can be compared

Appraisal of the Quality of Neurosurgery Clinical Practice Guidelines.

OBJECTIVE: The rate of neurosurgery guidelines publications was compared over time with all other specialties. Neurosurgical guidelines and quality of supporting evidence were then analyzed and compared by subspecialty. METHODS: The authors first performed a PubMed search for Neurosurgery and Guidelines. This was then compared against searches performed for each specialty of the American Board of Medical Specialties. The second analysis was an inventory of all neurosurgery guidelines published by the Agency for Healthcare Research and Quality Guidelines clearinghouse. All Class I evidence and Level 1 recommendations were compared for different subspecialty topics. RESULTS: When examined from 1970-2010, the rate of increase in publication of neurosurgery guidelines was about one third of all specialties combined (P \u3c 0.0001). However, when only looking at the past 5 years the publication rate of neurosurgery guidelines has converged upon that for all specialties. The second analysis identified 49 published guidelines for assessment. There were 2733 studies cited as supporting evidence, with only 243 of these papers considered the highest class of evidence (8.9%). These papers were used to generate 697 recommendations, of which 170 (24.4%) were considered Level 1 recommendations. CONCLUSION: Although initially lagging, the publication of neurosurgical guidelines has recently increased at a rate comparable with that of other specialties. However, the quality of the evidence cited consists of a relatively low number of high-quality studies from which guidelines are created. Wider implications of this must be considered when defining and measuring quality of clinical performance in neurosurgery

Partial purification and reconstruction of the Na⁺-D-glucose cotransport protein from pig renal proximal tubules

Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phospatidylserine by a freeze-thaw procedure. In the proteoliposomes Na+-D-glucose cotransport was demonstrated by showing that the D-glucose concentration in the liposomes increased far above the equilibrium value if a Na+ gradient was applied. The initial D-glucose uptake rate, stimulated by an inside directed gradient of 89 mM Na+, was 4 pmol/mg of protein-1 s-1. High affinity phlorizin binding could not be measured. After two precipitation steps with the solubilized membrane proteins, a protein fraction was obtained in which significantly high affinity phlorizin binding was detected. After reconstitution, proteoliposomes were formed in which more than 70% of the protein was represented by two polypeptides with molecular weights of 94,000 and 52,000. An initial Na+ gradient-dependent D-glucose uptake rate of 118 pmol/mg of protein-1 s-1 was obtained. In these liposomes, the D-glucose uptake rate could be inhibited by phlorizin (Ki = 0.3 μM), and 55-pmol phlorizin-binding sites per mg of protein (KD = 0.5 μM) were measured. In different liposomal preparations a correlation between Na+ gradient-dependent D-glucose uptake rate and the amount of 52,000 molecular weight polypeptide was observed