Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli

The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [3H]L-quinuclidinyl benzilate ([3H]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [(3)H]QNB with carbamylcholine and atropine. The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [(35)S]GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor

Land use inventory through merging of LANDSAT (satellite), aerial photography and map sources

There are no author-identified significant results in this report

A High-Order Unifying Discontinuous Formulation for the Navier-Stokes Equations on 3D Mixed Grids

This is the published version. Copyright 2011 © EDP SciencesThe newly developed unifying discontinuous formulation named the correction procedure via reconstruction (CPR) for conservation laws is extended to solve the Navier-Stokes equations for 3D mixed grids. In the current development, tetrahedrons and triangular prisms are considered. The CPR method can unify several popular high order methods including the discontinuous Galerkin and the spectral volume methods into a more efficient differential form. By selecting the solution points to coincide with the flux points, solution reconstruction can be completely avoided. Accuracy studies confirmed that the optimal order of accuracy can be achieved with the method. Several benchmark test cases are computed by solving the Euler and compressible Navier-Stokes equations to demonstrate its performance