Affinity enrichment of plasma membrane for proteomics analysis

Proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially a powerful approach for the identification of membrane-associated proteins responding to these environments. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. Here, we describe an affinity enrichment method, which combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads, for the isolation of plasma membranes. This method results in a 400-fold enrichment of plasma membrane relative to endoplasmic reticulum, a major contaminant in standard plasma membrane preparations, and dramatically reduces contamination from other cellular organelles. The biotinylation reaction did not interfere with ligand-dependent activation of receptor tyrosine kinases or G-protein coupled receptors, suggesting cell-surface signal transduction machinery remains functional. Membrane fractions prepared by this method should provide excellent starting materials for membrane proteomics analysis such as studies of dynamic trafficking and regulation of signaling molecules or identification of disease-specific membrane markers.     

Calculation of the Entropy of Lattice Polymer Models from Monte Carlo Trajectories

While lattice models are used extensively for macromolecules (synthetic polymers proteins, etc.), calculation of the absolute entropy, S, and the free energy, F, from a given Monte Carlo (MC) trajectory is not straightforward. Recently, we have developed the hypothetical scanning MC (HSMC) method for calculating S and F of fluids. Here we extend HSMC to self-avoiding walks on a square lattice and discuss its wide applicability to complex polymer lattice models. HSMC is independent of existing techniques and thus constitutes an independent research tool; it provides rigorous upper and lower bounds for F, which can be obtained from a very small sample and even from a single chain conformation.     

Synthesis of a homoleptic niobium(V) thiolate complex and the preparation of niobium sulfide via thio "sol-gel" and vapor phase thin-film experiments

Reaction of [Nb(NMe(2))(5)] with 10 equiv of 2,6-Me(2)C(6)H(3)SH in toluene results in the formation of red crystals of [Nb(SC(6)H(3)Me(2)-2,6)(5)]. Crystal structure analysis of [Nb(SC(6)H(3)Me(2)-2,6)(5)] showed that the niobium center adopts a distorted trigonal bipyramidal geometry. Niobium disulfide, NbS(2), has been successfully prepared via a thio "sol-gel" process using [Nb(SC(6)H(3)Me(2)-2,6)(5)] as the metal source. In contrast, vapor phase thin-film studies revealed that [Nb(SC(6)H(3)Me(2)-2,6)(5)] functions as a single-source precursor to NbS films.     

Synthetic, structural and spectroscopic studies of (pseudo)halo(1,3-di-tert-butylimidazol-2-ylidine)gold complexes

A series of (pseudo)halo(1,3-di-tert-butylimidazol-2-ylidine)gold complexes [(But2Im)AuX](X = Cl, Br, I, CN, N3, NCO, SCN, SeCN, ONO2, OCOCH3, CH3) have been synthesized and characterised spectroscopically and structurally. 13C NMR chemical shifts for the carbene carbon vary widely with differing ancillary anion, correlating well with the sigma-donor ability of the latter and with the M-C(carbene) bond distance. These results reinforce the notion that N-heterocyclic carbene ligands are primarily sigma-donor ligands with little pi-acceptor ability.     

Hydrocarbon oxidation vs C-C bond-forming approaches for efficient syntheses of oxygenated molecules

[Reaction: see text] A hydrocarbon oxidation approach has been applied to the construction of several linear (E)-allylic alcohols that have served as intermediates in the synthesis of natural products and natural product-like molecules. In the original syntheses, these intermediates were constructed using a standard Wittig-type olefination approach. We report here that routes to these same intermediates designed around a hydrocarbon oxidation approach are more efficient both in the total number of functional group manipulations (FGMs) and overall steps, as well as in the overall yield.     

Carbon-13 NMR spectra of rifamycins

The natural abundance ¹³C Fourier transform magnetic resonance spectra of rifamycin S and some of its derivatives have been studied. A combination of five different approaches has made unambiguous assignments for most of the resonances possible: (1) comparative study of the non- decoupled and noise-decoupled spectra; (2) ¹³C spectral characteristics; (3) spectral comparison between derivatives; (4) selective proton decoupling; (5) biogenetic evidence. Pulse and Fourier transform ¹³C NMR spectroscopy provides a more complete picture of these complex molecules than was previously obtained by ¹H NMR spectroscopy.     

Heat capacities of solid ortho—para mixtures of deuterium in the ordered state

The heat capacities of three ortho—para mixtures of solid deuterium in the rotationally ordered state as a function of temperature as well as the order—disorder transition temperature, T_c(x) for several compositions have been measured. The rotational heat capacities for all three mixtures, x (para or J = 1 mole fraction) = 0.69₉, 0.81₉ and 0.92₁ per mole of J = 1 species can be represented by a single function in terms of the reduced temperatures, T_c(x)/T, which to a good approximation is given by

, where Γ⁰_eff(1)/k = 1.0 deg is the effective electric quadrupolar coupling constant for the anisotropic interactions in pure para D₂ and T_c(1) = 4.05 degrees the order—disorder transition temperature. It is shown that this correlation follows from the assumption that both the libron energies and band widths of the libron modes scale with composition in a manner identical to the transition temperatures, namely

.     

Acoustic absorption in aqueous potassium phosphate

Measurements of acoustic absorption and velocity as a function of frequency and concentration in KH₂PO₄–K₂HPO₄ buffers at 4°C and pH 5-7 are reported. The dependence of the observed acoustic relaxation parameters on concentration is consistent with that to be expected from perturbation of a monomer-dimer equilibrium with an equilibrium constant [for 2H₂PO ₄ ^– (H₂PO₄)₂ ^2–] of 0.21 M^–1, a bimolecular rate constant of 5×10⁸ M^–1-sec^–1 and a standard volume change of –5 cm³ mole. The equilibrium constant for H₂PO ₄ ^– + HPO₄ ^2–H₃(PO₄)₂ ^3– is estimated to be 0.7 M^–1.     

Evaluation of the immobilized artificial membrane phosphatidylcholine: Drug discovery column for high-performance liquid chromatographic screening of drug–membrane interactions

Chromatographic retention factors (k′) of a series of eight β-adrenoceptor antagonist compounds (β-adrenolytic drugs) were determined employing an immobilized artificial membrane column (IAM.PC.DD). The influence of mobile phase pH, ionic strength, and organic modifier composition was studied in order to examine column performance. After the IAM.PC.DD columns were exposed to approximately 7000 column volumes of a 0.01 M PBS mobile phase, five out of six columns tested showed significant peak broadening and decreased k′ values indicative of premature column failure. The data suggested that the immobilized phospholipids stationary phase was removed by the 0.01 M PBS mobile phase. The β-adrenolytic drug's log k′_IAM values obtained with an IAM.PC.DD column were compared to an ^esterIAM.PC.MG column for predicting drug membrane interactions. For the linear regression analysis between log k′_IAM and the logarithm of the n-octanol–water partition coefficients (r_IAM.PC.DD=0.8710 vs. r_IAM.PC.MG=0.9538), the C₁₈ HPLC retention factors (r_IAM.PC.DD=0.8408 vs. r_IAM.PC.MG=0.9380), the liposome partition coefficients (r_IAM.PC.DD=0.8887 vs. r_IAM.PC.MG=0.9187), and various pharmacokinetic parameters, significantly better correlations were obtained with the ^esterIAM.PC.MG column than the IAM.PC.DD column.