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A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.

A chiral, divergent synthesis of two carbafuranosylamines, 1 and 2, two carbapyranosylamines, 3 and 4, two carbafuranosylamino acids, 5 and 6, and two carbapyranosylamino acids, 7 and 8, has been achieved. Highlights of the procedure include: a diastereoselective crossed vinylogous Mukaiyama aldol coupling between N-(tert-butoxycarbonyl)-2-ƒË(tert-butyldimethylsilyl)oxyƒÍpyrrole (TBSOP, 9) and 2,3-O-isopropylidene-D-glyceraldehyde (10) for the assembly of the target compound carbon backbone; a high yielding silylative cyclo-aldolization which gives the cyclopentanoid and cyclohexanoid motifs; and a reductive or hydrolytic breakage of the lactam C(O)¡VN link to liberate the carbasugar and install the desired pseudo-anomeric amine and the hydroxymethyl or carboxyl functionalities. The sequences leading to trans-configured carbafuranosyl compounds 1 and 5 and carbapyranosyl compounds 3 and 7 were 12 and 13 step processes, with overall yields of 34%, 35%, 17% and 16%. Cis-configured isomers 2, 4, 6, and 8 were obtained only in minor yields.

The silyloxy diene-based construction of carbasugars, previously exploited for the synthesis of four carbocyclic furanose and pyranose analogues, has been investigated further. By introducing a novel silylative cycloaldolization protocol and by adjusting a couple of minor transformations, the efficiency of this synthetic sequence was greatly improved. Through a series of lactone/thiolactone aldehyde cyclization precursors, four carbafuranoses (4a-carba-beta-D-xylofuranose, 4a-carba-beta-D-ribofuranose, 4a-carba-beta-L-arabinofuranose, and 4a-carba-beta-L-lyxofuranose) and four (carbafuranosyl)thiols [(4acarba-beta- D-xylofuranosyl)thiol, (4a-carba-beta-D-ribofuranosyl)thiol, (4a-carba-beta-L-arabinofuranosyl)- thiol, and (4a-carba-beta-L-lyxofuranosyl)thiol] were assembled. From this study, it was shown that these constructions tolerate a variety of precursors, and in many instances, they are suitable for scaling-up.

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

Spectroscopic non-targeted methods are gaining ever-growing importance in quality control and authenticity assessment of food products because of their strong potential for identification of specific features of the products by data-driven classifiers. One of the factors hampering the diffusion of spectroscopic non-targeted methods and data-driven classifiers is the lack of harmonized guidelines for their development and validation. In particular, to date, neither conditions to directly compare spectra recorded by different spectrometers nor studies demonstrating the statistical equivalence of the spectra are available. Among the spectroscopic analytical techniques suitable for the development of non-targeted methods, nuclear magnetic resonance (NMR) offers the unique opportunity to generate statistically equivalent signals. In this paper, the feasibility of NMR spectroscopy to generate statistically equivalent NMR signals from a number of different spectrometers was demonstrated for complex mixtures (aqueous extracts of wheat and flour) by organizing an inter-laboratory comparison involving 36 NMR spectrometers. Univariate statistics along with multivariate analysis were exploited to establish unbiased criteria for assessing the statistical equivalence of the NMR signals. The aspects affecting the signal equivalence were investigated, and possible solutions to reduce the extent of the human error were proposed and applied with satisfactory results. This study furnishes the scientific community with an appropriate and easy procedure to validate non-targeted NMR methods and provides error values to be used as a reference for future studies.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters. (Graph Presented).

Cultured HeLa cells were incubated with pyrene-GM1/3H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of 3H-GM1. The pyrene-GM1 and 3H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and 3H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GD1a) derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca2+ salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca2+ concentration dependent (maximum at 10 mM), and subsequent addition of EDTA had no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties.

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with [3H]acetic anhydride of very high specific radioactivity. The fluorenyl group removed by ammonia treatment was substituted by a nitrophenyl azide group. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 and cold natural GM1 (1:10 by mol) for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2 h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive: one, of about 30 kDa, interacted with the ganglioside molecules inserted into the outer membrane layer; three, in the region of 46 kDa, interacted with the portion of associated ganglioside able to be released by trypsin treatment. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins. When the incubation was prolonged to 24 h, photolabeled GM1 underwent extensive metabolic processing and gave origin to the corresponding ganglioside derivatives GM2, GM3, and GD1a. Under these conditions many proteins became radioactive, a consequence of GM1 transfer from the surface to the interior of the cell and of the ready availability of interactions of GM1 and of its metabolites. © 1989 American Chemical Society.

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [(Sph-3H]sulphatide), the second on C-1 of stearic acid [(stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.

N -Glycolylneuraminic acid containing GM1, GM1(NeuGc), was prepared by semisynthetic procedure. The procedure makes use of GM1 ganglioside deacetylated at the level of sialic acid residue (deAc-GM1) and of 1,3-dioxalan-2,4-dione. DeAc-GM1 is prepared from GM1 by alkaline hydrolysis in the presence of tetramethylammonium hydroxide and the glycolylating compound by reaction of glycolic acid with phosgene in dioxane, followed by cyclization under vacuum. Mass spectrometric and nuclear magnetic resonance spectroscopy analyses clearly indicated the presence, in the neosynthesized ganglioside of a glycolic group in the sialic acid residue. Laser-light scattering measurements show that GM1(NeuGc) aggregates in aqueous media being present in solution as micelles with a molecular weight of 576,000 and a hydrodynamic radius of 62.4 A as determined at 25°C. GM1 (NeuGc) promotes neurite outgrowth in N-2a cells to a similar degree as GM1 (NeuAc), but shows different behaviour under treatment with sialidase from Arthrobacter ureafaciens .

The thermotropic behavior (studied by high-sensitivity differential scanning calorimetry) and susceptibility to Vibrio cholerae sialidase hydrolysis of large unilamellar vesicles of dipalmitoyl-phosphatidylcholine, containing native GD1a ganglioside or the molecular species of GD1a containing C18:1 or C20:1 long-chain base (C18:1 GD1a; C20:1 GD1a), were studied. Vesicles containing ganglioside (10% in molar terms) showed the presence in the heat capacity function of a second minor peak besides the phospholipid main transition peak. The presence of a second peak is much more evident with C20:1 GD1a than with C18:1 GD1a, the difference being potentiated by Ca2+ and indicating a different tendency of the CD1a molecular species to undergo lateral phase separation. The scans of vesicles containing native GD1a showed the features of those obtained with C18:1 GD1a and C20:1 GD1a, indicating that the main components of native GD1a, C18:1 GD1a and C20:1 GD1a, maintain their individual aggregative properties. V. cholerae sialidase affects vesicle-bound GD1a at a much higher rate (17-25-fold) than it does micellar GD1a, the activation by Ca2+ being 3- and 2-fold, respectively. The Vmax values were identical on C18:1 GD1a and C20:1 GD1a in micellar dispersions, whereas they were markedly higher (from 20 to 50%) on C18:1 GD1a than on C20:1 GD1a in vesicular dispersions. Exhaustive sialidase hydrolysis of vesicles carrying native GD1a produced C18:1 GM1 and C20:1 GM1 in the same proportion as the C18:1 and C20:1 species present in native GD1a (53.9% and 46.1%).(ABSTRACT TRUNCATED AT 250 WORDS)