Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis

The ability of two-dimensional gel electrophoresis (2-DE) to separate glycoproteins was exploited to separate distinct glycoforms of kappa-casein that differed only in the number of O-glycans that were attached. To determine where the glycans were attached, the individual glycoforms were digested in-gel with pepsin and the released glycopeptides were identified from characteristic sugar ions in the tandem mass spectrometry (MS) spectra. The O-glycosylation sites were identified by tandem MS after replacement of the glycans with ammonia/aminoethanethiol. The results showed that glycans were not randomly distributed among the five potential glycosylation sites in kappa-casein. Rather, glycosylation of the monoglycoform could only be detected at a single site, T-152. Similarly the diglycoform appeared to be modified exclusively at T-152 and T-163, while the triglycoform was modified at T-152, T-163 and T-154. While low levels of glycosylation at other sites cannot be excluded the hierarchy of site occupation between glycoforms was clearly evident and argues for an ordered addition of glycans to the protein. Since all five potential O-glycosylation sites can be glycosylated in vivo, it would appear that certain sites remain latent until other sites are occupied. The determination of glycosylation site occupancy in individual glycoforms separated by 2-DE revealed a distinct pattern of in vivo glycosylation that has not been recognized previously.

Using two dimensional sectional distributions to infer three dimensional volumetric distributions - Validation using tomography

The critical process parameter for mineral separation is the degree of mineral liberation achieved by comminution. The degree of liberation provides an upper limit of efficiency for any physical separation process. The standard approach to measuring mineral liberation uses mineralogical analysis based two-dimensional sections of particles which may be acquired using a scanning electron microscope and back-scatter electron analysis or from an analysis of an image acquired using an optical microscope. Over the last 100 years, mathematical techniques have been developed to use this two dimensional information to infer three-dimensional information about the particles. For mineral processing, a particle that contains more than one mineral (a composite particle) may appear to be liberated (contain only one mineral) when analysed using only its revealed particle section. The mathematical techniques used to interpret three-dimensional information belong, to a branch of mathematics called stereology. However methods to obtain the full mineral liberation distribution of particles from particle sections are relatively new. To verify these adjustment methods, we require an experimental method which can accurately measure both sectional and three dimensional properties. Micro Cone Beam Tomography provides such a method for suitable particles and hence, provides a way to validate methods used to convert two-dimensional measurements to three dimensional estimates. For this study ore particles from a well-characterised sample were subjected to conventional mineralogical analysis (using particle sections) to estimate three-dimensional properties of the particles. A subset of these particles was analysed using a micro-cone beam tomograph. This paper presents a comparison of the three-dimensional properties predicted from measured two-dimensional sections with the measured three-dimensional properties.

Prediction of minimum spouting velocity in two-dimensional flat bottom spouted beds

Spouted beds have been used in industry for operations such as drying, catalytic reactions, and granulation. Conventional cylindrical spouted beds suffer from the disadvantage of scaleup. Two-dimensional beds have been proposed by other authors as a solution for this problem. Minimum spouting velocity has been studied for such two-dimensional beds. A force balance model has been developed to predict the minimum spouting velocity and the maximum pressure drop. Effect of porosity on minimum spouting velocity and maximum pressure drop has been studied using the model. The predictions are in good agreement with the experiments as well as with the experimental results of other investigators.

Erratum: Two-Dimensional Failure Modeling with Minimal Repair which appeared in this journal of April 2004, Volume 51:3 on pages 345–362

In this Erratum, we point out the reason for an error in the derivation of a result in our earlier paper, “Two-Dimensional Failure Modeling with Minimal Repair” [1], which appeared in the April 2004 issue of this journal, 51:3, on pages 345–362, and give the correct derivation.

NMR structure of C-terminally tagged gramicidin channels

A biotin group was covalently attached to the C terminus of gramicidin A (gA) through a linker arm comprising a glycine residue with either one (gAXB) or two caproyl groups (gAXXB). High-resolution two-dimensional NMR spectroscopy was used to determine the structure of these modified gA analogues and [Lys(16)]gramicidin A (gA-Lys) in sodium dodecyl-d(25) sulphate micelles. Gated gA ion channels based on linking a receptor group to these gA analogues have been used recently as a component in a sensing device. The conformations of the gA backbones and amino acid side chains of lysinated gA and biotinylated gA in detergent micelles were found to be almost identical to that of native gA, i.e. that of an N-terminal to N-terminal (head to head) dimer formed by two right-handed, single-stranded beta(6.3) helices. The biotin tail of the gAXB and gAXXB and the lysine extremity of gA-Lys appeared to lie outside the micelle. Thus it appears that the covalent attachment of functional groups to the C terminus of gA does not disrupt the peptide's helical configuration. Further, single channel measurements of all three gA analogues showed that functioning ion channels were preserved within a membrane environment. (C) 1999 Elsevier Science B.V. All rights reserved.

Solution structure of the sodium channel antagonist conotoxin GS: A new molecular caliper for probing sodium channel geometry

Background: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. Results: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. Conclusions: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

A nearly idealized 6 '-O-methylated t-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)

The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and C-13, H-1, and C-13/H-1 heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6-O-methylated iota-earrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported. (C) 2004 Elsevier Ltd. All rights reserved.

Reproducibility and accuracy of echocardiographic measurements of left ventricular parameters using real-time three-dimensional echocardiography

OBJECTIVES We sought to determine whether assessment of left ventricular (LV) function with real-time (RT) three-dimensional echocardiography (3DE) could reduce the variation of sequential LV measurements and provide greater accuracy than two-dimensional echocardiography (2DE). BACKGROUND Real-time 3DE has become feasible as a standard clinical tool, but its accuracy for LV assessment has not been validated. METHODS Unselected patients (n = 50; 41 men; age, 64 +/- 8 years) presenting for evaluation of LV function were studied with 2DE and RT-3DE. Test-retest variation was performed by a complete restudy by a separate sonographer within 1 h without alteration of hemodynamics or therapy. Magnetic resonance imaging (MRI) images were obtained during a breath-hold, and measurements were made off-line. RESULTS The test-retest variation showed similar measurements for volumes but wider scatter of LV mass measurements with M-mode and 2DE than 3DE. The average MRI end-diastolic volume was 172 +/- 53 ml; LV volumes were underestimated by 2DE (mean difference, -54 +/- 33; p < 0.01) but only slightly by RT-3DE (-4 +/- 29; p = 0.31). Similarly, end-systolic volume by MRI (91 +/- 53 ml) was underestimated by 2DE (mean difference, -28 +/- 28; p < 0.01) and by RT-3DE (mean difference, -3 +/- 18; p = 0.23). Ejection fraction by MRI was similar by 2DE (p = 0.76) and RT-3DE (p = 0.74). Left ventricular mass (183 +/- 50 g) was overestimated by M-mode (mean difference, 68 +/- 86 g; p < 0.01) and 2DE (16 +/- 57; p = 0.04) but not RT-3DE (0 +/- 38 g; p = 0.94). There was good inter- and intra-observer correlation between RT-3DE by two sonographers for volumes, ejection fraction, and mass. CONCLUSIONS Real-time 3DE is a feasible approach to reduce test-retest variation of LV volume, ejection fraction, and mass measurements in follow-up LV assessment in daily practice. (C) 2004 by the American College of Cardiology Foundation.