Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, β-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.     

Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.     

Role of sialic acids in rotavirus infection

Rotaviruses are the leading cause of childhood diarrhea. The entry of rotaviruses into the host cell is a complex process that includes several interactions of the outer layer proteins of the virus with different cell surface molecules. The fact that neuraminidase treatment of the cells, or preincubation of the virus with sialic acid-containing compounds decrease the infectivity of some rotavirus strains, suggested that these viruses interact with sialic acid on the cell surface. The infectivity of some other rotavirus strains is not affected by neuraminidase treatment of the cells, and therefore they are considered neuraminidase-resistant. However, the current evidence suggests that even these neuraminidase-resistant strains might interact with sialic acids located in context different from that of the sialic acids used by the neuraminidase-sensitive strains. This review summarizes our current knowledge of the rotavirus-sialic acid interaction, its structural basis, the specificity with which distinct rotavirus isolates interact with sialic acid-containing compounds, and also the potential use of these compounds as therapeutic agents.     

Presence of sialic acids in Lactobacillus plantarum

It was found that Lactobacillus plantarum (strain BA 11) is able to synthesize sialic acids during its growth in MRS medium and that these molecules are located mainly on the surface of the bacterium. It was demonstrated also that the addition externally of N-acetylneuraminic acid in concentrations ranged from 10 to 500 microM into the culture medium, resulted to a substantial increase of the growth rate of the bacterium. Bacterial cultures in presence of added sialic acid (100 microM) for 24 hours, resulted to a two fold increase of the final bacterial mass compared to the cultures in absence of sialic acid. Maximum levels of sialic acids were observed after 48 h of bacterial growth. It was also found that neuraminic acids production was increased when Mn++ and Mg++ ions were added in the culture medium, while the addition of Co++, Ca++, Ba++, Cu++ and Ni++ had a negative effect.     

Diversity of microbial sialic acid metabolism.

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.     

Fluorimetric assay of sialic acids.

A fluorimetric assay has been developed for sialic acids in which sialic acids react with pyridoxamine to give fluorescent compounds in the presence of zinc ion and pyridine. This assay method is specific for unbound sialic acids and is a simple and sensitive procedure compared with the thiobarbituric acid assay of sialic acids.     

The nature of sialic acids in human lymphocytes

Analysis of the sialic acids obtained by mild acid hydrolysis of B lymphocytes reveals the presence of N-acetylneuraminic acid and 9-O-acetyl-N-acetylneuraminic acid. For T lymphocytes only N-acetylneuraminic acid has been demonstrated to occur. The applied methods include quantitative colorimetry, thin-layer chromatography and combined gas-liquid chromatography-mass spectrometry.     

The participation of sialic acids in microglia-neuron interactions

Since it is known that sialic acid participates in neuronal plasticity, it is resonable to investigate its role in microglia-neuron interactions. In this study, we tested the effects of enzymatic removal of sialic acid on neurite and cell body density in microglia-neuron co-cultures. Additionaly, we analyzed the expression of Siglec-F protein, putative receptor for sialic acids, in microglial cells as well as its affinity to neurons. The results showed that removal of sialic acids affects neuronal integrity and changes microglial morphology. In presence of microglial cells, endoneuraminidase and α-neuraminidase significantly reduced neurite density (p<0.05). Endoneuraminidase (p<0.05) and α-neuraminidase (p>0.05) decreased the number of neuronal cell bodies in comparison to control co-cultures. Neuraminidases-treated neurons showed reduced binding of Siglec-F protein, which we found in microglial cells. Our results suggest that interactions between sialic acids and Siglec receptors may protect neuronal integrity during neurodegenerative processes.     

Histochemical analysis of sialic acids in the epididymis of the rat

 A variety of sialic acids contained in the rat epididymis were histochemically examined by means of lectin and pre-lectin methods by light microscopy. Epididymides from adult male Sprague-Dawley rats were fixed in Bouin’s fluid and routinely embedded in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Limax flavus, Sambucus nigra, Sambucus sieboldiana or Maackia amurensis lectins or to the selective periodate oxidation–phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The present results revealed that principal cells in the initial segment and caput contain sialic acid linked to α2,6-galactose/N-acetylgalactosamine, whereas those in the corpus and cauda include the sialic acidα2,3-galactose sequence. Narrow and clear cells involve all the types of sialic acids examined. Basal and halo cells mainly contain sialic acidα2,3-galactose. 8- And/or 9-O-acetylated sialic acids were predominantly distributed in principal cells of the initial segment and proximal caput. These findings are taken to indicate that various sialic acids in the epididymis could participate in different physiological functions characteristic of the regions in this organ. Accepted: 10 November 1997