The interaction of peripheral blood leukocytes with alpha1-acid glycoprotein, its carbohydrate chains and neoglycoconjugates

The interaction of human peripheral blood leukocytes with alpha 1-acid glycoprotein (AGP), its glycoforms as well as neoglyco-conjugates representing carbohydrate chains of AGP or its fragments was studied by flow cytometry. It was shown that the main target cells for AGP as well as for conjugates of its carbohydrate chains with polyacrylamide (PAA) are monocytes and polymorphonuclear leukocytes but not lymphocytes. The interaction of AGP with monocytes and granulocytes are mediated by its carbohydrate chains: the binding of AGP with cells was inhibited by AGP, AGP oligosaccharides as well as conjugates of oligosaccharides and its fragments with PAA. The data obtained show the existence of monocyte (and granulocyte) receptors which interact with complex type sialooligosaccharides of AGP.     

A new combined computational and NMR-spectroscopical strategy for the identification of additional conformational constraints of the bound ligand in an aprotic solvent

This study documents the feasibility of switching to an aprotic medium in sugar receptor research. The solvent change offers additional insights into mechanistic details of receptor--carbohydrate ligand interactions. If a receptor retained binding capacity in an aprotic medium, solvent-exchangeable protons of the ligand would not undergo transfer and could act as additional sensors, thus improving the level of reliability in conformational analysis. To probe this possibility, we first focused on hevein, the smallest lectin found in nature. The NMR-spectroscopic measurements verified complexation, albeit with progressively reduced affinity by more than 1.5 orders of magnitude, in mixtures of up to 50% dimethyl sulfoxide (DMSO). Since hevein lacks the compact beta-strand arrangement of other sugar receptors, such a structural motif may confer enhanced resistance to solvent exchange. Two settings of solid-phase activity assays proved this assumption for three types of alpha- and/or beta-galactoside-binding proteins, that is, a human immunoglobulin G (IgG) subfraction, the mistletoe lectin, and a member of the galectin family of animal lectins. Computer-assisted calculations and NMR experiments also revealed no conspicuous impact of the solvent on the conformational properties of the tested ligands. To define all possible nuclear Overhauser effect (NOE) contacts in a certain conformation and to predict involvement of exchangeable protons, we established a new screening protocol applicable during a given molecular dynamics (MD) trajectory and calculated population densities of distinct contacts. Experimentally, transferred NOE (tr-NOE) experiments with IgG molecules and the disaccharide Gal'alpha1-3Galbeta1-R in DMSO as solvent disclosed that such an additional crosspeak, that is, Gal'OH2--GalOH4, was even detectable for the bound ligand under conditions in which spin diffusion effects are suppressed. Further measurements with the plant lectin and galectins confirmed line broadening of ligand signals and gave access to characteristic crosspeaks in the aprotic solvent and its mixtures with water. Our combined biochemical, computational, and NMR-spectroscopical strategy is expected to contribute notably to the precise elucidation of the geometry of ligands bound to compactly folded sugar receptors and of the role of water molecules in protein--ligand (carbohydrate) recognition, with relevance to areas beyond the glycosciences.     

Study of sialated neoglycoconjugates by surface enhanced Raman scattering spectroscopy

Neoglycoconjugates based on polyacrylamide and sialic acid with N-acetylneuraminic acid or sialooligosaccharides as side chains were studied by surface-enhanced Raman scattering (SERS) spectroscopy. It had previously been found that these polymers can effectively inhibit influenza virus adhesion. This study revealed the possibility to evaluate, based on the intensity of SERS signals, the overall availability for interaction and the conformational freedom of sialic acid residues in glycoconjugates. The dependence of these two factors on the structure and density of sialylated side chains was studied. The uniformity of distribution of sialylated side chains in conjugates was shown. Comparison of the results of the SERS spectroscopic study of the conjugates and the data on their inhibitory effect on the adhesion of specific strains of influenza virus allowed the identification of the conjugates for which the availability and conformational freedom of sialic acid are the main factors determining their inhibitory properties. A conclusion was also reached about the predominance of one of the mechanisms (competitive inhibition or steric stabilization) in the inhibitory properties of the specific conjugates.     

Surface enhanced Raman spectroscopy for characterization of structural characteristics of carbon chains in alpha1-acid glycoprotein and pseudoglycoproteins]

Surface-enhanced Raman scattering (SERS) spectroscopy was used to study the structure of carbohydrate chains in glycosylated forms of alpha 1-acid glycoprotein (AGP) and in pseudoglycoproteins obtained by transferring the carbohydrate chains of AGP to a polyacrylamide carrier. It was found that AGP-D glycoform and pseudoglycoproteins containing three or more glycans per molecule, which possess high immunomodulating activity, have a specific spatial organization of carbohydrate chains. This organization is maintained by the interaction of neighboring glycans with each other and does not depend on the nature of the carrier (whether it is polypeptide or polyacrylamide).     

Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells

The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.     

Recent achievements in the development of partitioning processes of minor actinides from nuclear wastes obtained in the frame of the NEWPART European programme (1996–1999)

Partitioning of long-lived minor actinides (americium and curium) from the nuclear wastes issuing the reprocessing of nuclear spent fuels, in order to transmute them into short-lived nuclides or to condition them into stable crystalline matrices, was the subject of intense research within the NEWPART research program of the European 4^th Frame Work Program, FWP (1996–1999). The target waste considered was the acidic raffinate (HAR) issuing the reprocessing of the used nuclear fuels by the PUREX process. A two step separation process based on liquid-liquid extraction was designed. The first step consists in the co-separation of the mixture of trivalent actinides and lanthanides from the HAR by extraction with a malonamide extractant (DIAMEX process), while the second step concerns the actinides(III)/lanthanides(III) group separation (SANEX process). Several DIAMEX and SANEX processes were developed and successfully tested with cold, spiked and genuine high active effluents. The research carried out also included basic and fundamental works in order to better understand the relationships between the structures of the extractants and their affinities for the target metal ions. The lecture highlighted both the basic and applied aspects of the research. This work will be pursued (PARTNEW program) within the 5^th FWP of the European Union during the period 2000–2003.     

Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion

BACKGROUND: Human natural xenoantibodies represent a major hurdle to the clinical application of pig lungs in transplantation by initiating hyperacute rejection within minutes to hours. OBJECTIVE: The object was to compare pig organ perfusion and specific depletion of anti-alpha-galactosyl xenoantibodies for prevention of hyperacute rejection in the pig to human lung combination. METHODS: Large White pig (20-25 kg) left lungs were removed and continuously ventilated and reperfused ex vivo either with (1) whole human blood previously perfused in situ through pig right lung (group I), liver (group II), or spleen (group III) or with (2) human plasma in vitro immunoabsorbed on columns containing alpha-galactosyl disaccharide (Gal-alpha-(1-3)Gal-beta-(CH2)3NH2; B disaccharide) (group IV). Each study group included 6 animals. RESULTS: The in situ and in vitro preperfusions depleted anti-alpha-galactosyl xenoantibodies and all in situ perfused pig organs showed histologic signs of hyperacute rejection. After the ex vivo reperfusion, group I xenografts had a significantly (P < .001) longer functional and histologic survival than did xenografts in groups II, III, and IV. Human blood reperfusing group I xenografts had a significantly (P < 0.05) lower (1) decline of clotting factors and total circulating immunoglobulins, (2) total and membrane attack complex (C5b,6,7,8,9) complement activation, and (3) hemolysis. By Western blot analysis, the in situ lung preperfusion removed antibodies against non-alpha-galactosyl proteins of low molecular weight that were not eliminated by the alpha-galactosyl column. CONCLUSIONS: Results demonstrate that specific depletion of anti-alpha-galactosyl antibodies alone incompletely protects pig lungs from hyperacute rejection. It is speculated that the more complete prevention of this rejection afforded by pig lung preperfusion relates to the removal of other, non-alpha-galactosyl antibodies.     

Cohesion energy densities and solubility parameters for the n-alkanes expressed as a new homomorph equation

A new correlation formula for the calculation of the cohesive energy density for n-alkanes and the corresponding homomorphs for polar solvents has been developed. Constants of the Antoine equation and literature values of heat of vaporisation at the boiling point and at 25°C were used. For the C₂-C₁₆ n-alkanes the new correlation equation describes the cohesion energy density as a function of molal volume and reduced temperature within the accuracy of experimental data. In the range of reduced temperatures 0.4 < T_r < 0.7 and molal volumes 50 < V <300 cm³ the maximum deviation for this fit is less than ± 0.05 J^1/2 cm^?3/2. In the region of T_r and V for polar homomorphs to the n-alkanes at 25°C the deviations are typically less than half this value.     

On the Synergy Effect in MoO3–Fe2(MoO4)3 Catalysts for Methanol Oxidation to Formaldehyde

Methanol oxidation to formaldehyde was studied over a series of Fe–Mo–O catalysts with various Mo/Fe atomic ratio and the end compositions Fe₂O₃ and MoO₃. The activity data show that the specific activity passes through a maximum with increase of the Mo content and is the highest for Fe₂(MoO₄)₃. The selectivity to formaldehyde, on the other hand, increases with the Mo content in the catalyst. A synergy effect is observed in that a catalyst with the Mo/Fe ratio 2.2 is almost as active as Fe₂(MoO₄)₃ and as selective as MoO₃. Imaging of a MoO₃/Fe₂(MoO₄)₃ catalyst by SEM and TEM shows that the two phases form separate crystals, and HRTEM reveals the presence of an amorphous overlayer on the Fe₂(MoO₄)₃ crystals. EDS line-scan analysis in STEM mode demonstrates that the Mo/Fe ratio in the amorphous layer is ~2.1 in the fresh catalyst and ~1.7 in the aged catalyst. The enrichment of Mo at the catalyst surface is confirmed by XPS data. Raman spectra give evidence for the Mo in the amorphous material being in octahedral coordination, which is in contrast to the crystalline Fe₂(MoO₄)₃ bulk structure where Mo has tetrahedral coordination. X-ray diffraction (XRD) analysis gives no support for the formation of a defective molybdate bulk structure. The results presented give strong support for the Mo rich amorphous structure being observed on the Fe₂(MoO₄)₃ crystal surfaces being the active phase for methanol oxidation to formaldehyde.