Gangliosides and sialic acid effects upon newborn pathogenic bacteria adhesion: An in vitro study

The effect of the main gangliosides (GM₁, GM₃, GD₃) and free sialic acid (Neu5Ac) upon the adhesion of pathogenic bacteria implicated in infant diarrhoea is assessed in vitro using the Caco-2 cell line. Concentrations of the bioactive compounds found in the bioaccessible (soluble) fraction of infant formula and human milk are employed. Bacterial adhesion behaviour included enterotoxigenic Escherichia coli (ETEC), enteropathogenic E.coli (EPEC), Listeria monocytogenes, Salmonella entericaserovartyphi, Shigella sonnei, Campylobacter jejuni and Helicobacter pylori. Three different approaches were assayed: pre-incubation of bacteria and compounds before addition to cells (competition); pre-incubation of the cells with compounds (exclusion); and pre-incubation of cells with bacteria (displacement). Furthermore, the spatial localization of the most abundant gangliosides, GM₃ and GD₃, in Caco-2 cells has been determined using confocal microscopy. Results show that GM₃, GD₃, GM₁ and Neu5Ac at the assayed concentrations are able to interfere with the adhesion of several pathogenic bacteria involved in neonatal diseases-the greatest effect corresponding to Neu5Ac, followed by GD₃, GM₁ and GM₃. Gangliosides GM₃ and GD₃ are located in the apical and basolateral membranes of the Caco-2 cells.     

Fabrication of GM3-Enriched Sphingomyelin/Cholesterol Solid-Supported Lipid Membranes on Au/SiO2 Plasmonic Substrates

The fabrication of solid-supported artificial lipid bilayers enriched with biological agents is an important step for acquiring more insights into their behavior from in vitro studies. In this work we demonstrate the formation of lipid artificial membranes enriched with ganglioside GM3, whose role in the cell proliferation and tumor angiogenesis is still an object of extensive investigation. The membranes are formed by fusion of small unilamellar vesicles (SUV) obtained from the sonication of multi-lamellar vesicles (MLV) formed from a hydrated mixture of GM3, brain sphingomyelin and cholesterol. The effective formation of SUV was confirmed by Dynamic light scattering (DLS) measurements. The recognition of the ganglioside in the biomimetic raft like biomembranes is accomplished via surface plasmon resonance (SPR) by exploiting the ganglioside affinity with wheat germ agglutinin (WGA). The affinity constant of the binding between the inglobated GM3 and WGA has been measured for three different GM3 molar concentrations. Beside the effective generation of GM3-enriched biomimetic membranes, our results indicate a decrease in the apparent WGA–GM3 dissociation constant at increasing GM3 concentrations up to 20 mol%, consistent with clustering effects of the ganglioside in the fabricated biomembranes.     

Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers

Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross‐sectional sample of Chinese mothers over an 8‐month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast‐feeding period of 1?6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM₃ molecular species containing long‐chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long‐chain acyl fatty acid GD₃ molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long‐chain acyl fatty acid molecular species were more varied. The major GM₃ and GD₃ molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.     

Milk ingredients as nutraceuticals

Cow's milk is considered as a basic food in many diets, and is rich in a variety of essential nutrients. With today's sophisticated analytical, biochemical and cell-biological research tools, the presence of many other (minor) compounds with biological activity has been demonstrated. Achievements in separation techniques in the dairy industry and enzyme technology offer opportunities to isolate, concentrate or modify these compounds, so that their application in functional foods, dietary supplements, nutraceuticals and medical foods has become possible.
Within the sequence of amino acids of a dietary protein, specific peptides may be located with specific biological activity. Peptides from casein may be used to enhance the solubility of minerals such as calcium and zinc—and hence increase the bioavailability of these minerals—or to reduce the activity of the angiotensin-converting enzyme, which is involved in vasoconstriction and (hence) blood pressure.
Immunoglobulins from vaccinated cows may be considered as natural antimicrobials with certain advantages over synthetic antibiotics. Lactoferrin is an example of a minor milk protein that has been studied in great detail: it is becoming increasingly clear that it is important for the nonspecific defence against bacteria, fungi and viruses. Oligosaccharides, glycolipids and glycoproteins containing sialic acid residues may have a role as anti-infectives.
Components such as growth factors may also be considered for future product development, because of the economies of scale used in the dairy industry.     

The β-Subunit of Cholera Toxin has a High Affinity for Ganglioside GM1 Embedded into Solid Supported Lipid Membranes with a Lipid Raft-Like Composition

In this communication, we report on the fabrication of GM1-rich solid-supported bilayer lipid membranes (ssBLM) made of sphingomyelin and cholesterol, the main components of lipid rafts, which are the physiological hosting microenvironment of GM1 on the cell membrane. The functionality of the ganglioside has been checked by measuring the apparent dissociation constant K _D of the complex formed by the β-subunit of the cholera toxin and GM1. The value found deviates less than one order of magnitude from that measured for in vivo cells, indicating the potential of these ssBLM as optimized in vitro biomimetic platforms.     

Comparative Analysis of Brain Lipids in Mice,Cats, and Humans with Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid (GSL) storage disease that arises from an autosomal recessive mutation in the gene for the β-subunit of β-Hexosaminidase A (Hexb gene), which catabolizes ganglioside GM2 within lysosomes. Accumulation of GM2 and asialo-GM2 (GA2) occurs primarily in the CNS, leading to neurodegeneration and brain dysfunction. We analyzed the total lipids in the brains of SD mice, cats, and humans. GM2 and GA2 were mostly undetectable in the normal mouse, cat, and human brain. The lipid abnormalities in the SD cat brain were generally intermediate to those observed in the SD mouse and the SD human brains. GM2 comprised 38, 67, and 87% of the total brain ganglioside distribution in the SD mice, cats, and humans, respectively. The ratio of GA2–GM2 was 0.93, 0.13, and 0.27 in the SD mice, cats, and humans, respectively, suggesting that the relative storage of GA2 is greater in the SD mouse than in the SD cat or human. Finally, the myelin-enriched lipids, cerebrosides and sulfatides, were significantly lower in the SD brains than in the control brains. This study is the first comparative analysis of brain lipids in mice, cats, and humans with SD and will be important for designing therapies for Sandhoff disease patients.