Lactational changes in the fatty acid composition of human milk gangliosides

The objectives of this work were to study the FA composition of milk gangliosides, as well as to gain further insight into the characterization of human milk gangliosides. The potential capacity of human milk gangliosides to adhere to human enterotoxigenic Escherichia coli (ETEC-strains) was also studied. Human milk gangliosides were isolated and identified by high-performance TLC or immunoassay. The latter also was used to assay bacterial adhesion. The FA composition of gangliosides was studied by GC. The presence of O-acetyl GD3 (Neu5,9Ac₂α2–8 NeuAcα2–3Galβ1–4GlcCer) and trace amounts of GM1 [Galβ1–3GalNAcβ1,−3(NeuAcα2–3)Galβ1–4GlcCer] in human milk was confirmed. Medium-chain FA were almost absent in colostrum, whereas in the subsequent stages they rose to 20%. The levels of long-chain FA decreased after colostrum. With respect to the degree of saturation, gangliosides from colostrum were richer in monounsaturated FA than gangliosides synthesized during the rest of the lactation period, opposite to the pattern for PUFA. A human-ETEC colonization factor antigen II-expressing strain showed binding capacity to human milk GM3 (NeuAcα2–3Galβ1–4GlcCer). New data on human milk gangliosides have been gathered. A thorough knowledge of their composition is needed since they may have important biological implications in regard to newborns' defense against infection. The ganglioside nomenclature of Svennerholm (34) is followed.     

Bovine milk gangliosides: Changes in ceramide moiety with stage of lactation

The stage of lactation is one of the most important factors that influence milk composition. Changes in fatty acids from triacylglycerols and phospholipids have already been reported. In this study, we looked for a lactational change in the ganglioside lipid moeity since ganglioside contents and patterns vary strongly with stage of lactation. Individual gangliosides from four stages were isolated, methanolyzed to cleave the bonds between individual constituents, and derivatized for gas-liquid chromatography and gas chromatography/mass spectrometry analyses. Ceramide components, both fatty acids (as methyl esters derivatives) and long-chain bases, were identified and quantified. The results pointed to a marked change in ceramide from colostrum to milk that was characterized by a dramatic decrease in saturated and the longest-chain fatty acids as well as an increase in 18∶1 and 18∶2. The major long-chain base along lactation was a recently described structure, 3-ethoxy-15∶0 sphinganine. Other new long-chain base structures appeared in these gangliosides. All these changes suggest differences in the fluidity of the fat globule membrane, reflecting physiological variations in cows with respect to milk production.     

The Antiadhesive Strategy in Crohn′s Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent‐invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn′s disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg^?1) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water‐soluble and non‐cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.     

Microbial Biofouling: A Mechanistic Investigation

Microbial biofouling is important in a variety of applications including reverse osmosis membranes and in-situ bioremediation. The initial microbial adhesion plays the key role in microbial biofouling on abiotic surfaces, which can be explained in terms of microbial surface properties. This research investigated the interactions of three indigenous bacteria with the porous medium of silica sand and their corresponding initial attachment. Traditional and extended DLVO forces were calculated based on independently measured bacterial and medium surface thermodynamic properties and were related to bacterial attachment observations. Lifshitz–van der Waals, electrostatic and Lewis acid/base forces were found to be strongly dependent on the separation distance between bacteria and the medium surface. It was concluded that the electrostatic force served as the barrier preventing the bacterial strains from getting close to the medium. Once the bacterial strains overcame the barrier with the aid of hydrodynamic forces, Lifshitz–van der Waals force and electrostatic force dropped while Lewis acid/base force increased with the decrease of separation distance. Consequently, Lewis acid/base force became the dominating force controlling bacterial adhesion.     

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.     

Die Zusammensetzung tierischer Organ- und Depotfette in Abhängigkeit von der Fütterung II Gas-chromatographische Analyse der Fettsäuren von Kälber- und Rinderfetten

The Composition of Lipids of Animal Organ and Depot Fats in Relation to Feed II: Gas-chromatographic Analysis of Fatty Acids of Calves and Cattle Fats In the milk as well as in the fats of calves and cattle 81 fatty acids were found. The composition of fatty acids of the depot and organ fats of cattle — in winter with stall-feeding and in summer with pasture-feeding — were compared with the winter and summer milk fats. The differences between the fats from the same parts of calves and cattle are discussed.     

Longitudinal Evolution of the Concentration of Gangliosides GM3 and GD3 in Human Milk

It has been reported that dietary gangliosides may have an important role in preventing infections and in brain development during early infancy. However, data related to the evolution of their concentration over the different stages of lactation are scarce. Liquid chromatography coupled with electrospray ionization high resolution mass spectrometer (LC/ESI‐HR‐MS) has been optimized to quantify the two major ganglioside classes, i.e., aNeu5Ac(2‐8)aNeu5Ac(2‐3)bDGalp(1‐4)bDGlcp(1‐1)Cer (GD3) and aNeu5Ac(2‐3)bDGalp(1‐4)bDGlcp(1‐1)Cer (GM3) in human milk. Gangliosides were extracted using chloroform and methanol, further purified by solid‐phase extraction and separated by reversed‐phase liquid chromatography. Repeatability, intermediate reproducibility, and recovery values were assessed to validate the method. In human milk, GD3 and GM3 could be quantified at the level of 0.1 and 0.2 μg/mL, respectively, with relative standard deviation of repeatability [CV(r)] and intermediate reproducibility [CV(iR)] values ranging from 1.9 to 15.0 % and 1.9 to 22.5 %, respectively. The described method was used to quantify GD3 and GM3 in human milk samples collected from 450 volunteers between 0 and 11 days and at 30, 60 and 120 days postpartum, providing for the first time the concentration of these minor lipids in a large cohort. The content of total gangliosides ranged from 8.1 and 10.7 μg/mL and the mean intake of gangliosides in infants 30, 60 and 120 days postpartum could be estimated at about 5.5, 7.0 and 8.6 mg of total gangliosides per day, respectively, when infants were exclusively breastfed.     

Squaric Acid Monoamide Mannosides as Ligands for the Bacterial Lectin FimH: Covalent Inhibition or Not?

Bacteria use long proteinaceous appendages, called fimbriae or pili, to adhere to the surfaces of their host cells. Widely distributed among the Enterobacteriacae are type 1 fimbriae that mediate mannose-specific bacterial adhesion through the lectin FimH, located at the fimbrial tips. It is possible to design synthetic mannosides such that they show high affinity for FimH and can thus inhibit mannose-specific bacterial adhesion in a competitive manner. It has been found that mannosidic squaric acid monoamides serve especially well as inhibitors of type 1 fimbriae-mediated bacterial adhesion, but it has remained unclear whether this effect is due to specific inhibition of the bacterial lectin FimH or to unspecific bioconjugation between the lectin's carbohydrate binding site and a squaric acid monoamide. A bioconjugation reaction would result in a covalently crosslinked squaric acid diamide. Here it is shown that covalent inhibition of FimH by mannosidic squaric acid derivatives is very unlikely and that compounds of this type serve rather as excellent specific candidates for low-molecular-weight inhibitors of bacterial adhesion. This has been verified by testing the properties of glycosidic squaric acid monoamides in diamide formation, by two different adhesion assays with a series of selected control compounds, and by molecular docking studies that further support the results obtained in the bioassays.     

Relevance of Cell Wall and Extracellular Matrix Proteins to Staphylococcus Epidermidis Adhesion and Biofilm Formation

In the present study, the protein profiles of the cell wall (CW) extracts of eight Staphylococcus epidermidis strains were analysed. The protein pattern of the extracellular matrix (EM) of four S. epidermidis good biofilm producer strains was also obtained. The main goal was to relate these protein patterns with their bacterial adhesion and biofilm forming ability. For this purpose, 2 h adhesion and 8 d-old biofilm assays were carried out, with the adhered cells and biomass quantified by microscopic observation and crystal violet quantification, respectively. CW and EM proteins were visualized by SDS–PAGE. According to the results, a significant percentage of the CW proteins detected was common to all the strains studied. However, the most adhering strains expressed a high number of proteins associated with the initial adhesion process, and the strongest biofilm producers expressed proteins that were absent in the protein profile of the strains that produced lower amounts of biofilm. Therefore, the present results demonstrate the importance of such proteins in adhesion and biofilm formation processes. Some of the CW proteins were also present in the EM protein pattern, though to a smaller amount, as well as several exoenzymes important for the host immune system invasiveness.     

Simulation of the attachment system with various tip shapes contacting rough surface

In this paper, we investigate the effects of various contact shapes on the adhesion performance for the attachment system contacting rough surfaces. Four different tip shapes of sphere, flat punch, torus and tape are considered. We demonstrate the effect of the tip size and the elastic modulus on the adhesion force through the adhesion analysis for single tips with four different shapes. In order to investigate an effect of tip shape on adhesion performance for the attachment system contacting rough surface with different σ values, single level attachment system was simulated. The attachment system is modeled from the geometrical size of spatulae of Tokay gecko. The effect of tip shape on the adhesion enhancement and how far its effect can be reached for the surface roughness are investigated. It is shown that the effect of tip shape on the adhesion enhancement is limited to the surface roughness with smaller than several micrometers and further adhesion enhancement can not be expected at rougher surfaces.     

Gangliosides and atherosclerosis

The ganglioside levels in atherosclerotic lesions of human aorta are considerably higher than those in unaffected areas of aorta, and atherosclerotic patients frequently have increased concentrations of serum gangliosides. The present review summarizes recent findings that suggest the possible involvement of aortic gangliosides in platelet activation and adhesion of platelets to the vessel wall. The effect of gangliosides on the structure of low density lipoproteins (LDL), on the interaction of LDL with macrophages and hepatic cells and on the LDL-regulated biosynthesis of cholesterol is also discussed.In vitro experiments have demonstrated that a major ganglioside of the intima of atherosclerotic aorta induces rapid adhesion, aggregation and spreading of platelets. Moreover, gangliosides present in elevated amounts in the intercellular space of atherosclerotic aortic tissue modify the surface structure and stimulate aggregation of LDL. Ganglioside-modified LDL are readily recognized and taken up by macrophages, while preincubation of LDL with low concentrations of gangliosides inhibits LDL binding to hepatic cells. Thus, ganglioside enrichment of LDL is likely to interfere with LDL clearancevia the hepatic cells. Thus, ganglioside enrichment of LDL is likely to interfere with LDL clearancevia the hepatic LDL receptor, and to stimulate binding of LDL to the scavenger receptor of macrophages. It is postulated that high ganglioside levels in the aorta and serum may be an additional risk factor in atherosclerosis. The abbreviations used for gangliosides are standard abbreviations as recommended by Svennerholm [Svennerholm, L. (1964)J. Lipid Res. 5, 145–155].     

Distribution of glycosphingolipids of monkey small and large intestinal mucosa

The ganglioside and neutral glycosphingolipid composition of adult monkey small and large intestinal mucosa were characterized and compared. GM₃, GM₂ and GD_1A were found to be the principal gangliosides in each of these tissues. Dihexosylceramide was the major neutral glycosphingolipid of both organs. The total content of gangliosides and neutral glycolipids/ceramide, however, was ca. four-fold and two-fold higher, respectively, in small intestinal than colonic mucosa. While all glycosphingolipids examined contained hydroxy and nonhydroxy fatty acids, the former fatty acids accounted for 60–90% of the total fatty acids in both organs. Sphingosine was the predominant long chain base of ceramide, mono-, di-, tri- and tetrahexosylceramide, whereas phytosphingosine was the major base of GM₃ in both tissues. The results of these studies demonstrate that while many similarities of monkey small and large intestinal glycosphingolipids exist, qualitative and quantitative differences are present along the length of the monkey gut. These differences may be at least partially responsible for certain of the well-recognized variations in normal physiological and pathological processes that occur in these organs.     

Escherichia coli Biofilm Characteristics on Polymeric Heat Exchanger Surfaces

Time‐dependent effects on the apparent roughness and surface free energy of different polymeric surfaces and stainless steel were studied during the biofouling process for Escherichia coli K12. The surface roughness increases during primary adhesion of E. coli on the surfaces and is later reduced as the surface between scattered bacteria is completely covered, forming a uniform biofilm. During the fouling process, the polar fraction of the surface free energy significantly increased, whereas the dispersive fraction decreased for all substrates. The attachment of E. coli and subsequent bacterial production of extracellular polymeric substances increased the polarity of the initially nonpolar polymeric surfaces to increase wettability.     

Systematic analysis of glycosphingolipids in the human gastrointestinal tract: Enrichment of sulfatides with hydroxylated longer-chain fatty acids in the gastric and duodenal mucosa

The composition of the glycosphingolipids of the human gastrointestinal tract was studied. The major neutral glycosphingolipids were ceramide monohexosides (e.g., GalCer, GlcCer), LacCer, Gb₃Cer, Gb₄Cer and more polar ones with more than four sugars, whereas neither Gg₃Cer nor Gg₄Cer were present. The acidic glycosphingolipids consisted of sulfatides and gangliosides such as G_M3, G_M1, G_D3 and G_D1a. Also a large amount of sulfatides was found in the gastric mucosa and duodenum. The concentrations of sulfatides in the fundic mucosa, antral mucosa and duodenum amounted to 416.0, 933.8 and 682.9 nmol/g of dry weight, respectively, exceeding those in the gastric mucosa and kidney of other mammals. The major molecular species of the sulfatides were identified as I³SO₃-GalCer with hydroxylated longer-chain fatty acids based on the analyses by gas-liquid chromatography and negative ion fast-atom bombardment mass spectrometry. In contrast, gangliosides in these regions showed a tendency to be lower than sulfatides, and the molar ratios of sulfatides to gangliosides were about 2.0, whereas those in other parts were less than 0.5. A high content of sulfatides in the gastric and duodenal mucosa, where mucosa is easily insulted by acid, pepsin and bile salts, may be closely related to their roles in mucosal protection. The nomenclature used for gangliosides and other glycosphingolipids follows the system of Svennerholm (Ref. 1) and the recommendation of the IUPAC-IUB Commission (Ref. 2), respectively.     

Polyunsaturated fatty acid supply with human milk

Polyunsaturated fatty acids in human milk may derive from diet, liberation from maternal body stores, or endogenous synthesis from precursor fatty acids. The contribution of each of these sources has not been studied in detail. Although maternal diet is a key factor affecting human milk composition, other factors such as gestational age, stage of lactation, nutritional status, and genetic background are known to influence the fat content and fatty acid composition in human milk. Both linoleic and α-linolenic acids, the essential fatty acids, are present in human milk, as are several other n−6 and n−3 longer chain polyunsaturated fatty acids that are required for optimal growth and development of infants. The fatty acid profile of human milk from lactating women of different countries is remarkably stable, but there is variability in some of the components, such as docosahexaenoic acid, which is mainly due to differences in dietary habits. Tracer techniques with stable isotopes have been valuable in assessing the kinetics of fatty acid metabolism during lactation and in determining the origin of fatty acids in human milk. Based on these studies, the major part of polyunsaturated fatty acids in human milk seems not to be provided directly from the diet but from maternal tissue stores.     

Triacylglycerol structure of human colostrum and mature milk

Because triacylglycerol (TAG) structure influences the metabolic fate of its component fatty acids, we have examined human colostrum and mature milk TAG with particular attention to the location of the very long chain polyunsaturated fatty acid on the glycerol backbone. The analysis was based on the formation of various diacylglycerol species from human milk TAG upon chemical (Grignard degradation) or enzymatic degradation. The structure of the TAG was subsequently deduced from data obtained by gas chromatographic analysis of the fatty acid methyl esters in the diacylglycerol subfractions. The highly specific TAG structure observed was identical in mature milk and colostrum. The three major fatty acids (oleic, palmitic and linoleic acids) each showed a specific preference for a particular position within milk TAG: oleic acid for thesn-1 position, palmitic acid for thesn-2 position and linoleic acid for thesn-3 position. Linoleic and α-linolenic acids exhibited the same pattern of distribution and they were both found primarily in thesn-3 (50%) andsn-1 (30%) positions. Their longer chain analogs, arachidonic and docosahexaenoic acids, were located in thesn-2 andsn-3 positions. These results show that polyunsaturated fatty acids are distributed within the TAG molecule of human milk in a highly specific fashion, and that in the first month of lactation the maturation of the mammary gland does not affect the milk TAG structure.     

人产肠毒素大肠杆菌基因工程疫苗的研究进展

产肠毒素大肠杆菌(ETEC)是引起腹泻的主要致病菌之一,在世界范围内,每年可导致数亿人发生腹泻,尤其是发展中国家的婴幼儿和儿童。目前,世界各国通过构建新型的ETEC基因工程疫苗预防ETEC引起的腹泻。将ETEC的肠毒素基因和菌毛蛋白基因转化至无毒的受体菌中,构建出基因工程疫苗,免疫人体后可有效预防ETEC引起的腹泻。本文就ETEC的致病机理及国内外ETEC基因工程疫苗的最新研究进展作一综述。     

Activation of soil respiration and shift of the microbial population balance in soil as a response to Lavandula stoechas essential oil

Lavandula stoechas, a native plant of Greece, is rich in essential oil and fenchone is its major constituent. We examined the effect of the essential oil and its main constituents on soil metabolism and microbial growth. Addition of the essential oil or fenchone to soil samples induced a remarkable increase in soil respiration. This was accompanied by an increase in the soil bacterial population of three orders of magnitude. This sizable population was not qualitatively similar to that of the control soil samples. One bacterial strain dominated soil samples treated with L. stoechas essential oil or fenchone. By use of the disk diffusion assay, we evaluated the capacity of three bacterial strains that we isolated from the soil samples, as well as Escherichia coli and Bacillus subtilis (reference strains), to grow in the presence of the essential oil and three of its main constituents (fenchone, cineol, -pinene). The substances tested did not inhibit the growth of the strain found to dominate the bacterial populations of treated soil samples; they severely inhibited B. subtilis. The other two isolated strains could also grow in liquid cultures in the presence of different quantities of essential oil or fenchone. Addition of fenchone at the end of the exponential phase increased the cell numbers of the strain that dominated the bacterial populations of treated soil samples, indicating use of the substrate added. On the basis of these results, we propose a scheme of successional stages during the decomposition process of the rich-in-essential-oil litter of aromatic plants that abound in the Mediterranean environment.