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Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1–75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Gram-negative Campylobacter jejuni pathogenic in humans but commensal in chickens. [•] Cell surface glycosylation is a virulence factor which may alter with temperature. [•] Surface glycosylation of two strains compared between human and avian temperatures. [•] LPS-like and N-linked structures dominant at 37°C and LPS reduced for 81116 at 42°C. [•] Relative distribution of CPS and LOS structures altered for 81-176 at 42 from 37°C. [ABSTRACT FROM AUTHOR]

Abstract: Vibrio parahaemolyticus is a seafood-borne pathogen which causes acute inflammatory gastroenteritis – a process which is mediated by the translocation of type three secretion system effector proteins. The molecular interactions governing colonization of the intestinal epithelium by this pathogen remain poorly understood. The mannose-sensitive haemagglutinin (MSHA) pilus was identified in this study as a significant factor in bacterial-host cell adherence and subsequent pathogenesis towards Caco-2 human intestinal epithelial cells. Deletion of essential components of the MSHA pilus resulted in a 60% decrease in adherence and a similar reduction in bacterial uptake by human intestinal cells. The diminished adherence of MSHA mutants correlated with significant decreases in V. parahaemolyticus-induced Caco-2 cell lysis, cell rounding and IL-8 secretion. Glycan array comparison between the V. parahaemolyticus wild type and MSHA deficient mutants identified lectin functionality for the MSHA pilus with specificity towards the fucosylated blood group oligosaccharide antigens Lewis A and X and blood groups A and B. The MSHA pilus also exhibited high affinity for the structurally related asialo-GM1 ganglioside, lacto-N-fucopentaose I and lacto-N-difucohexaose I. We hypothesize that these glycans act as receptors for the MSHA pilus in the gastrointestinal tract, thereby facilitating efficient colonization of the intestinal epithelium by V. parahaemolyticus. [Copyright &y& Elsevier]

Summary: Mucin-type O-glycosylation has been well characterized in mammalian systems but not in plants. In this study, the purified alcohol-soluble, non-reduced protein (prolamin) fraction from rice seed was investigated for the occurrence of O-linked oligosaccharides. As storage prolamins are unlikely to be O-glycosylated, any O-glycosylation found was likely to belong to co-extracted proteins, whether because of association with the protein body or solubility. SDS-PAGE and MS analyses revealed 14 and 16kDa protein families in fractions that bound to the lectins peanut agglutinin (PNA), Vicia villosa lectin (VVL) and Jacalin, indicative of the presence of O-linked saccharides. Enzymatic cleavage, fluorescent labeling and high-performance liquid chromatography (HPLC) analysis demonstrated a peak consistent with Gal-β-(1→3)-GalNAc, with similar MS/MS fragmentation. Additionally, upon chemical analysis, a GlcNAc-containing O-linked carbohydrate moiety was discovered. Protein blotting with anti-O-GlcNAc antibody (clone CTD110.6) was positive in a subpopulation of the 14kDa alcohol-soluble protein fraction, but a hot capping experiment was negative. Therefore, the GlcNAc residue in this case is unlikely to be terminal. Additionally, a positive reaction with CTD110.6mAb cannot be taken as absolute proof of O-GlcNAc modification and further confirmatory experiments should be employed. We hypothesize that O-glycosylation may contribute to protein functionality or regulation. Further investigation is required to identify the specific proteins with these modifications. This ‘reverse’ approach could lead to the identification of proteins involved in mRNA targeting, signaling, translation, anchoring or maintenance of translational quiescence and may be applied to germinating rice seed extracts for further elucidation of protein function and regulation. [Copyright &y& Elsevier]

Abstract: The structure of the phenol-soluble polysaccharide from Pseudoalteromonas rubra type strain ATCC 29570T has been elucidated using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, gNOESY, ROESY, 1H,13C gHMQC and gHMBC experiments. It is concluded that the trisaccharide repeating unit of the polysaccharide has the following structure: ▪ where Sug is 2-acetamido-2,6-dideoxy-d-xylo-hexos-4-ulose, Am is acetimidoyl and Acyl is a malic acid residue, which is O-acetylated in ∼70% of the units. [Copyright &y& Elsevier]

Abstract: An acidic O-specific polysaccharide (PS) of the agar-digesting bacterium Shewanella japonica with the type strain KMM 3299T was obtained by mild acid hydrolysis of the lipopolysaccharide. The polysaccharide was studied by component analysis, methylation analysis, 1H and 13C NMR spectroscopy, including 2D NMR experiments. The PS was determined to have the following structure involving three unusual amino sugars: [Copyright &y& Elsevier]

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of the marine bacterium Shewanella fidelis type strain KMM 3582T and studied by sugar analysis along with 1H and 13C NMR spectroscopy including one-dimensional NOE in difference mode and two-dimensional experiments. The polysaccharide was found to consist of linear tetrasaccharide repeating units containing N#x03B5;-[(S)-1-carboxyethyl]-Nα-(d-galacturonoyl)-l-lysine and having the following structure:The amide of d-galacturonic acid with N#x03B5;-[(S)-1-carboxyethyl]-l-lysine (`alaninolysine'', 2S,8S-AlaLys) was found for the first time in nature as a component of the O-specific polysaccharide of Providencia rustigianii O14 (Carbohydr. Res. 2003, 338, 1009–1016). [Copyright &y& Elsevier]

Mild acid degradation of the lipopolysaccharide of the bacterium Idiomarina zobellii, type strain KMM 231T, with aq 2% HOAc at 100 °C, yielded an oligosaccharide, which represents one repeating unit of the O-polysaccharide. A polysaccharide was obtained by mild base degradation of the lipopolysaccharide. The following structure of the O-polysaccharide was elucidated by 1H and 13C NMR spectroscopy of the oligosaccharide and base-degraded lipopolysaccharide, including COSY, TOCSY, ROESY, 1H, 13C HSQC, HSQC-TOCSY and HMBC experiments:→ 3)-α-d-Quip4N-(1 → 4)-α-d-GlcpA-(1 → 6)-α-d-GlcpNAc-(1 → 4)-α-l-GulpNA-(1 → 3)-β-d-FucpNAc-(1 →The O-polysaccharide is distinguished by the presence of two unusual amino sugars, 4-amino-4,6-dideoxy-d-glucose (d-Qui4N) and 2-amino-2-deoxy-l-guluronic acid (l-GulNA), both having the free amino group. The unexpectedly high acid lability of the glycosidic linkage of 2-acetamido-2,6-dideoxy-d-galactose (d-FucNAc) could be associated with the presence of a free amino group adjacent to the site of attachment of FucNAc to Qui4N. [Copyright &y& Elsevier]

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,13C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3′-carboxamide-2′,2′-dihydroxypropyl)-2,6-dideoxy-d-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:PS1→8)-α-8eLegp5Am7Ac-(2→3)-β-Shef-(1→PS2→8)-α-8eLegp5Am7Ac-(2→3)-β-Shep-(1→Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2. [Copyright &y& Elsevier]

The bovine milk fat globule membrane (MFGM) is a source of food-derived glycans that can offer an approach to prevent Escherichia coli O157:H7 infection by inhibiting attachment of the pathogen to host cells. Such glycans may decrease the need for antibiotic treatment by acting as prophylactics. In this study, we generated a defatted bovine MFGM fraction, rich in proteins and glycoproteins, and demonstrated its ability to prevent the association of several enterohaemorrhagic E. coli O157:H7 strains with human colonic adenocarcinoma, HT-29 cells. This defatted MFGM fraction reduced bacterial association with HT-29 cells in a concentration dependent and strain specific manner. This study may present a new approach to mitigate the adverse health effects caused by E. coli infections in humans. [ABSTRACT FROM AUTHOR]

The bovine milk glycoprotein bovine lactoferrin (bLF) has a variety of biological activities related to its constituent glycans. However, little is known about bLF's oligosaccharide structural changes over the course of lactation. BLF was isolated at 13 time points during the first three months of lactation from three individual cows and glycosylation changes were profiled by lectin microarrays. Substantial profile differences between early and late lactation were observed and accompanying monosaccharide analysis revealed that the occurrence of the non-human sialic acid, N-glycolylneuraminic acid, was greater during early stage milk production. Overall, the data suggested that more diverse complex-type oligosaccharide structures were present on bLF during early lactation with an abundance of oligomannose type glycans in later lactation. The differences in the glycoprofiles of bLF from colostrum to mature milk suggest that these may have different functionality in vivo. [ABSTRACT FROM AUTHOR]

The bovine milk fat globule membrane (MFGM) has many associated biological activities, many of which are linked with specific carbohydrate structures of MFGM glycoconjugates. Bovine buttermilk is a commercially viable source of MFGM and is an under-valued by-product of butter making. However, the changes in buttermilk glycosylation over the course of lactation have not been extensively investigated. In this study, buttermilk was generated from three individual multiparous cows at 13 time points over the first three months of lactation. Buttermilk glycosylation was profiled using lectin microarrays and lectin blotting. Suggested differences in glycosylation, including N-glycosylation, sialylation and fucosylation, were observed between early and late time points and between individual animals. Overall, these data suggest temporal changes in the glycosylation of buttermilk proteins which may have an important impact on commercial isolation of glycosylated ingredients. [ABSTRACT FROM AUTHOR]

[Display omitted] • Lectin microarray profiling showed detailed glycan compositions of WPC ingredients. • Enriched whey protein concentrate (WPC) ingredient glycosylation differed from WPC. • WPC global glycosylation of a MFGM-enriched WPC was similar to α-lactalbumin-WPC. • N -linked glycans of MFGM-WPC and α-lactalbumin-WPC were equivalent. • WPC ingredient protein profiles did not correlate with global glycosylation. Human milk fat globule membrane (MFGM) and whey proteins are nutritionally and functionally valuable, with many beneficial bioactivities associated with their glycosylation. However glycosylation of milk components other than free milk oligosaccharides are underinvestigated. Whey protein concentrate (WPC) ingredients with various enrichments or depletions are used in infant formula (IF) formulations to contribute to human milk equivalence and bioactivity benefits, but their overall or global glycosylation has not been compared. We compared the global glycosylation of commercial WPC ingredients for use in various IF formulations; two MFGM-enriched WPC ingredients (high fat HF1 and lower fat HF2), an α-lactalbumin-enriched WPC (WPC Lac) which has α-lactalbumin concentration closer to human milk and significantly less β-lactoglobulin which is not present in human milk, and two base WPC ingredients (WPC 80 and WPC 35) using lectin microarray profiling. WPC Lac and WPC HF1 glycosylation were highly similar to each other and both somewhat similar to WPC 35, while WPC HF2 was more similar to the base WPC 80 ingredient. N -linked glycosylation analysis demonstrated that WPC HF1 and WPC Lac were qualitatively most similar to one another, with WPC 80 and WPC 35 having similar structures, confirming lectin microarray profiling as a valuable method to compare global glycosylation. Thus WPC Lac may be a valuable ingredient for providing equivalent glycosylation to MFGM supplementation. [ABSTRACT FROM AUTHOR]

Aeromonas (A) gum, an extracellular heteropolysaccharide produced by the bacterium Aeromonas nichidenii strain 5797, was studied by 1H and 13C NMR spectroscopy including 2D COSY, TOCSY, 1H, 13C HMQC, HMBC and ROESY experiments after O-deacetylation and Smith degradation. These investigations revealed the presence of an O-acetylated pentasaccharide repeating unit composed of mannose, glucose, xylose and glucuronic acid, and it has the following structure: [Copyright &y& Elsevier]

Six polysaccharides were extracted sequentially from the fresh sclerotium of Poria cocos cultivated in China using 0.9% NaCl (PCS1), hot water (PCS2), 0.5M NaOH (PCS3-I and PCS3-II), and 88% formic acid (PCS4-I and PCS4-II). Their chemical and physical characteristics were determined using infrared spectroscopy (IR), gas chromatography (GC), GC–MS methylation analysis, 13C NMR spectroscopy, elementary analysis (EA), protein analysis, size exclusion chromatography combined with laser light scattering (SEC-LLS), light scattering (LS), and viscometry. The results indicated that the polysaccharides PCS1, PCS2, and PCS3-I were heteropolysaccharides containing d-glucose, d-galactose, d-mannose, d-fucose, and d-xylose; the predominant monosaccharide was d-glucose except for PCS1 where it was d-galactose. PCS3-II, the main component of the sclerotium of P. cocos, was a linear (1→3)-β-d-glucan of high purity. PCS4-I consisted of (1→3)-β-d-glucan with some β-(1→6) linked branches. PCS4-II was mainly composed of (1→3)-β-d-glucan containing some glucose branches. The Mw values of the six polysaccharides PCS1, PCS2, PCS3-I, PCS4-I in 0.2M NaCl aqueous solution, PCS3-II, and PCS4-II in dimethyl sulfoxide (Me2SO) were determined to be 11.6×104, 20.8×104, 17.1×104, 9.1×104, 12.3×104, and 21.1×104, respectively. The six polysaccharides in aqueous solution or Me2SO exist as flexible chains. [Copyright &y& Elsevier]

The chemical structures of polysaccharides and LPS core oligosaccharides, isolated from various Gram-negative marine bacteria from the genera Pseudoalteromonas and Shewanella belonging to the Alteromonadaceae family and γ-subclass of Proteobacteria, are reviewed. The polysaccharides are distinguished by the acidic character (e.g., due to the presence of hexuronic and aldulosonic acids and their derivatives) and the occurrence of unusual sugars, including N-acyl derivatives of 6-deoxyamino sugars, such as N-acetyl-d-quinovosamine, N-acetyl-l-fucosamine and N-acetyl-6-deoxy-l-talosamine, and higher sugars like 2,6-dideoxy-2-acetamido-4-C-(3′-carboxamide-2′,2′-dihydroxypropyl)-d-galactopyranose (shewanellose). Many constituent sugars have various uncommon non-sugar substituents, such as alanine, formic, lactic and hydroxybutyric acids, sulfate, phosphate, and 2-aminopropane-1,3-diol. [Copyright &y& Elsevier]

Critical limb ischemia (CLI) is characterized by the impairment of microcirculation, necrosis and inflammation of the muscular tissue. Although the role of glycans in mediating inflammation has been reported, changes in the glycosylation following muscle ischemia remains poorly understood. Here, a murine CLI model was used to show the increase of high mannose, α-(2, 6)-sialic acid and the decrease of hybrid and bisected N-glycans as glycosylation associated with the ischemic environment. Using this model, the efficacy of an elastin-like recombinamers (ELR) hydrogel was assessed. The hydrogel modulates key angiogenic signaling pathways, resulting in capillary formation, and ECM remodeling. Arterioles formation, reduction of fibrosis and anti-inflammatory macrophage polarization wa also induced by the hydrogel administration. Modulation of glycosylation was observed, suggesting, in particular, a role for mannosylation and sialylation in the mediation of tissue repair. Our study elucidates the angiogenic potential of the ELR hydrogel for CLI applications and identifies glycosylation alterations as potential new therapeutic targets. [ABSTRACT FROM AUTHOR]