Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.     

The Fascinating Chemistry of α-Haloamides

The aim of this review is to highlight the rich chemistry of α-haloamides originally mainly used to discover new C−N, C−O and C−S bond forming reactions, and later widely employed in C−C cross-coupling reactions with C(sp³), C(sp²) and C(sp) coupling partners. Radical-mediated transformations of α-haloamides bearing a suitable located unsaturated bond has proven to be a straightforward alternative to access diverse cyclic compounds by means of either radical initiators, transition metal redox catalysis or visible light photoredox catalysis. On the other hand, cycloadditions with α-halohydroxamate-based azaoxyallyl cations have garnered significant attention. Moreover, in view of the important role in life and materials science of difluoroalkylated compounds, a wide range of catalysts has been developed for the efficient incorporation of difluoroacetamido moieties into activated as well as unactivated substrates.     

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting

Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.     

Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea β-Vignin during Seed Germination

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein β-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of β-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. β-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.     

Atomic force microscopy study of lymphoblastoid cells under 50-Hz 2-mT magnetic field irradiation

Received: 18 December 1997/Accepted: 5 January 1998     

Thermoelectric power of p-doped single-wall carbon nanotubes and the role of phonon drag

We measured thermoelectric power S of bulk single-wall carbon nanotube materials p doped with acids. In contrast to oxygen-exposed or degassed samples, S is very small at the lowest temperatures, increases superlinearly above a characteristic and sample-dependent T, and then levels off. We attribute this unusual behavior to 1D phonon drag, in which the depression of the Fermi energy cuts off electron-phonon scattering at temperatures below a characteristic T0. This idea is supported by a model calculation in which the low temperature behavior of phonon drag is specifically related to the one-dimensional character of the electronic spectrum.     

Photoelectron spectra of 1,4-dithiin and related compounds

The photoelectron (He(I)) spectra of 1,4-dithiin (1), 1,4-thiaselenin (2), 1,4-thiatellurin (3) and 4H-thiopyran (4) were assigned by comparison with published spectra of similar compounds and with the help of ab initio and semi-empirical calculations. The proposed orbital sequences for the outermost orbitals are π₄, π₃, π₂, σ for 1, 2 and 3, and π₄, π₂, σ, π₃ for 4. π₄ and π₃ correspond to the symmetric and antisymmetric combinations, respectively, of the heteroatom lone pairs, while π₂ is the antisymmetric combination of the π_eth orbitals.     

Full automation of serotonin determination by column-switching and HPLC

Summary A simple, fast, fully automated method for plasma serotonin determination is described. Full automation is obtained by coupling two devices: a sample processing station and a solid-phase autosampler. The sample processing station dilutes the plasma sample and is then connected, on-stream, with the solid-phase autosampler. It firstly fills a loop with all the solvents necessary for the sample clean-up, then, inverting the flow, pumps these solvents through the silica-bonded cation-exchange disposable extraction cartridge positioned on the autosampler. For the elution, the cartridge is switched on-stream with the HPLC analytical column and serotonin is eluted by the HPLC mobile-phase. The HPLC separation is performed by ion-pairing reversed-phase liquid chromatography. The column effluent is completely reduced by an electrochemical reactor and serotonin is detected in an oxidation-mode by a dual-cell electrochemical detector. The plasma sample is 50 l, the plasma sensitivity is 40 ng/l, the retention time is 6 min and the recovery is 95%. The repeatibility, the normal ranges for platelet-poor and for platelet-rich plasma have been established and correlation with manual HPLC calculated.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Carbon-13 n.m.r. and CNDO/S study of 1,2-indandione and heterocyclic analogues

¹³C n.m.r. spectra are reported for 1,2-indandione, coumarandione and thionaphthenequinone in CDCl₃ solution and for isatin in dimethyl sulphoxide solution. The chemical shifts are assigned and discussed in connection with the influence of the heteroatom on the dicarbonyl group and the aromatic ring. The ¹³C shieldings have also been calculated by means of Pople's perturbation theory using CNDO/S wave functions.     

Some Properties of GMRES in Hilbert Spaces

Our purpose in this work is to explore the properties of GMRES in Hilbert spaces. We extend to the infinite dimensional context some main results that are known to hold in the finite dimensional case. A key assumption for these extensions is that the involved linear operator is an algebraic operator.