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This article describes the development, optimization, and evaluation of groundnut oil-entrapped tamarind seed polysaccharide (TSP)-alginate floating beads containing diclofenac sodium by emulsion-gelation method for gastroretentive delivery. The effect of polymer to drug ratio, groundnut oil to water ratio, and sodium alginate to TSP ratio on drug entrapment, density, and drug release of floating beads was optimized using three-factor and two-level factorial design. The optimized floating beads showed drug entrapment efficiency of 82.48% ± 2.34%, density of 0.88 ± 0.07 g/[cm.sup.3], cumulative drug release of 41.02% ± 0.86% after 8 h and floated well over 8 h in simulated gastric fluid (pH 1.2) with 8.25 min floating lag-time. The in vitro drug release from these floating beads containing diclofenac sodium was followed controlled-release pattern (zero-order kinetics) with case-II transport mechanism. The average bead size was ranged from 1.07 ± 0.03 to 1.94 ± 0.09 mm. The floating beads were characterized by SEM, FTIR, and [.sup.1]H NMR. The optimized floating beads showed excellent anti-inflammatory activity in carrageenan-induced rats over prolonged period after oral administration. POLYM. ENG. SCI., 53:238-250, 2013. © 2012 Society of Plastics Engineers, INTRODUCTION Oral controlled release drug delivery systems have been developed over the past few years due to their considerable therapeutic benefits such as ease of administration, patient compliance, and flexibility [...]

Ammonium-based alkali-catalyzed [beta]-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with [beta]1,3-1inked cores. In contrast to the previously studied [beta]1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the [beta]l,3-linkaga is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages ([alpha]l,2-, [beta]1,2-, [alpha]l,3-,[beta]1,3-, [alpha]l,4-, [beta]1,4-, [alpha]1,6-, and [beta]l,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O-and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O-and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glyean release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies. 10.1021/ac102300r

In this article, we report a novel lectin-based biosensor for electrochemical assay of cancer-associated glycosylation by comparative study of mannose and sialic acid expression on normal and cancer cells derived from human lung, liver, and prostate. Using a sandwich format, high sensitivity and selectivity were achieved by combining the lectin-based biosensor with the {lectin-Au-Th} bioconjugates featuring lectin and thionine (Th) labels linked to gold nanoparticles (AuNPs) for signal amplification. The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells as compared to normal ones. The results were in good agreement with those from fluorescent microscopy studies. The differences in the two glycan expression indicated that sialic acid could serve as a potential biomarker for early cancer detection. The lectin-based biosensor was also successfully used to quantify cancer cells and evaluate the average amount of sialic acid on single cell surface, which could supply significant information on glycan functions in cancer progression. Overall, the lectin-based electrochemical biosensor provides an effective pathway to analyze glycan expression on living cells and may greatly facilitate the medical diagnosis and treatment in early process of cancer. 10.1021/ac102132p

Due to its fastness and its easiness to use, MALDI-MS is currently an analytical tool widely used in glycomic applications. However, the MALDI ionization process could result in the so-called 'in-source decay', or ISD, of analytes, leading to complex spectra. On the other hand, ISD opens the possibility to perform pseudo-[MS.sup.3] experiments. This phenomenon must therefore be controlled in order to be used on demand as a supplementary tool for the analysis of permethylated glycans by MALDI mass spectrometry. For this purpose, several matrices were tested and MALDI imaging was used to determine optimal conditions promoting or, inversely, avoiding ISD of permethylated glycans. 2,5-DHB was shown to be a versatile matrix allowing one to induce or prevent ISD according to the location of laser shots. Inversely, it was shown that 9-aminoacridine forms homogeneous spots and avoids completely ISD. This matrix would therefore be suitable for automatic analysis. 10.1021/ac1017452

Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, [SCC.sub.15305RM] and [SCC.sub.15305cap]. The [SCC.sub.15305cap] element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK ([capD.sub.Ssp], [capL.sub.Ssp], and [capK.sub.Ssp]) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and [capI.sub.Ssp] show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by [sup.1]H and [sup.13]C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: -4)-[beta]-Glc-(1-3)-Sug-(1-4)-[beta]-GlcA-(1-[beta]-GlcNAc-(1-2) Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists. doi: 10.1128/JB.00104-10

The analysis of N-linked glycans by mass spectrometry (MS) has been characterized by low signal-to-noise ratios and high limits of detection due to their hydrophilicity and lack of basic sites able to be protonated. As a result, every step in glycan sample preparation must be thoroughly optimized in order to minimize sample loss, contamination, and analytical variability. Importantly, properties of glycans and their derivatized counterparts must be thoroughly studied in order to exploit certain characteristics for enhancing MS analysis. Herein, the effectiveness of the incorporation of a permanent charge is studied and determined to hamper glycan analysis. Also, a procedure for glycan hydrazone formation is optimized and outlined where a large number of variables were simultaneously analyzed using a fractional factorial design (FFD) in order to determine which conditions affected the reaction efficiency of the hydrazone formation reaction. Finally, the hydrophobic tagging of glycans is shown to be a viable opportunity to further increase the ion abundance of glycans in MS. 10.1021/ac101227a

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then be released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method. 10.1021/ac1011377

An improved approach for the surface modification of poly(dimethylsiloxane) (PDMS) using carboxymethyl cellulose (CMC), carboxymethyi [beta]-1,3-dextran (CMD), and alginic acid (AA) was investigated. The PDMS substrates were first oxidized in a H2SO4/H202 solution to transform the Si--C[H.sup.3] groups on their surfaces into Si--OH groups. Then methacrylate groups were grafted onto the substrates through a silanization reaction using 3-(trimethoxysilyl)propyl methacrylate. Sequentially, cysteamine was conjugated onto the sllanized surfaces by the reaction between the thiol and methacrylate groups under 254 nm UV exposure. Afterward, the amino-terminated PDMS substrates were sequentially reacted with CMC, CMD, and AA in the presence of N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide, resulting in the grafting of polysaccharides onto PDMS surfaces. The composition and chemical state of the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS). In addition, the stability and dynamic characteristics of the polysaccharide-grafted PDMS substrates were investigated by XPS and temporal contact angle experiments. A protein adsorption assay using bovine serum albumin (BSA), chicken egg albumin, lysozyme, and RNase-A showed that the introduction of CMD and AA can reduce the adsorption of negatively charged BSA and chicken egg albumin, but increase the adsorption of the positively charged lysozyme and RNase-A. However, CMC-modified PDMS surfaces showed protein-repelling properties, regardless of whether the protein was positively or negatively charged. A cell culture and migration study of glioma C6, MKN-45, MCF-7, and HepG-2 cells revealed that the polysaccharide-modified PDMS greatly improved the cytocompatibility of native PDMS. 10.1021/ac100544x

The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/ solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously described reductive [beta]-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals. 10.1021/ac100131e

Glycosylation often mediates important biological processes through the interaction of carbohydrates with complementary proteins. Most chemical tools for the functional analysis of glycans are highly dependent upon various linkage chemistries that involve the reducing terminus of carbohydrates. However, because of ring opening, the structural integrity of the reducing sugar ring (pyranose or furanose) is lost during these techniques, resulting in derivatized carboydrates that markedly differ from the parent molecule. This paper describes a new aqueous-based, one-pot strategy that involves first converting the sugar to a C-glycoside ketone, followed by conversion to ketohydrazones or oximes. Hence, the C-glycoside ketones are tagged with fluorescence, colored, cationic or biotin-labeled groups or immobilized onto hydrazine-functionalized beads. No activating or protecting groups are required, and the chemistry is mild enough for a wide range of carbohydrates. We demonstrate the versatility of the approach to diverse glycans, including bead immobilization and lectin analysis of acarbose, an antidiabetic drug, to dabsyl-tagged enzyme substrates to screen cellulases, and for the analysis of plant cell wall hemicellulosics. 10.1021/ac902894u

Fast, sensitive, robust methods for 'high-level' glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid 'high-level' glycan screening. 10.1021/ac902617t

Negative ion mode nanoliquid chromatography/mass spectrometry (nano-LC/MS) on porous graphitic carbon columns at pH 11 was studied and compared to capillary LC/MS at pH 8 for the analysis of neutral and acidic glycan alditols. Oligosaccharides were chromatographed with an acetonitrile gradient containing 0.04% ammonium hydroxide and analyzed with a linear ion trap mass spectrometer (LTQ) equipped with a modified nanospray interface. Analysis of acidic N- and O-glycan standards revealed that good quality MS/MS spectra could be obtained when loading 1-3 fmol, a 10-fold increase in sensitivity compared to capillary-LC/MS at pH 8. Analysis of a complex mixture of O-glycans from porcine colonic mucins with nano-LC/MS and MS/MS at high pH revealed 170 oligosaccharides in one analysis, predominantly corresponding to sulfated glycans with up to 11 residues. Analysis of the same sample with capillary-LC/MS showed a lower sensitivity for multiply sulfated glycans. Nano-LC/MS of O-linked oligosaccharides on MUC2 from a human colon biopsy also illustrated that the ionization of oligosaccharides with multiple sialic acid groups was increased compared to those with only one sialic acid residue. Nano-LC/MS at high pH is, thus, a highly sensitive approach for the analysis of acidic oligosaccharides. 10.1021/ac902602e

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.09.010 Byline: Jacopo F. Novelli (a), Kshitiz Chaudhary (a), Julie Canovas (a), Jack S. Benner (a), Catherine L. Madinger (a), Paul Kelly (c), Jonathan Hodgkin (b), Clotilde K.S. Carlow (a) Keywords: C. elegans; Galactofuranose; UGM; Surface coat; Glycans; M. nematophilum; Nematodes Abstract: Galactofuranose (Gal.sub.f), the furanoic form of d-galactose produced by UDP-galactopyranose mutases (UGMs), is present in surface glycans of some prokaryotes and lower eukaryotes. Absence of the Gal.sub.f biosynthetic pathway in vertebrates and its importance in several pathogens make UGMs attractive drug targets. Since the existence of Gal.sub.f in nematodes has not been established, we investigated the role of the Caenorhabditis elegans UGM homolog glf-1 in worm development. glf-1 mutants display significant late embryonic and larval lethality, and other phenotypes indicative of defective surface coat synthesis, the glycan-rich outermost layer of the nematode cuticle. The glf homolog from the protozoan Leishmania major partially complements C. elegans glf-1. glf-1 mutants rescued by L. major glf, which behave as glf-1 hypomorphs, display resistance to infection by Microbacterium nematophilum, a pathogen of rhabditid nematodes thought to bind to surface coat glycans. To confirm the presence of Gal.sub.f in C. elegans, we analyzed C. elegans nucleotide sugar pools using online electrospray ionization-mass spectrometry (ESI-MS). UDP-Gal.sub.f was detected in wild-type animals while absent in glf-1 deletion mutants. Our data indicate that Gal.sub.f likely has a pivotal role in maintenance of surface integrity in nematodes, supporting investigation of UGM as a drug target in parasitic species. Author Affiliation: (a) Division of Molecular Parasitology, New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA (b) Department of Biochemistry, Genetics Unit, South Parks Road, OX1 3QU Oxford, UK (c) Salem State College, 352 Lafayette Street, Salem, MA 01970, USA Article History: Received 30 March 2009; Revised 15 August 2009; Accepted 7 September 2009

N-Glycosylation of proteins is recognized as one of the most common post-translational modifications. It was believed that N-glycosylation occurred exclusively in eukaryotes until the recent discovery, of the general protein glycosylafion pathway (Pgl) in Campylobacter jejuni, which has similarities to the eukaryotic system and adds proteins en bloc from a lipid carrier to a protein acceptor. In addition to N-linked glycans, a number of pathogenic bacteria such as Pseudomonas aeruginosa and Neisseria species have been shown to O-glycosylate their proteins through polyisoprene-linked intermediates. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. With the increasing frequency of reports of bacterial protein glycosylation that proceed through lipid-mediated steps, there is a need for technologies capable of characterizing these newly described bacterial systems as well as eukaryotic pathways from biologically relevant samples in an accurate, rapid, and cost-effective manner. In this paper, a new glycomics strategy based on porous graphite carbon (PGC) liquid chromatography mass spectrometry (LC-MS) was devised and validated on the C. jejuni N-glycan pathway, lipid-linked oligosaccharide intermediates of the Pgl pathway from crude lipid extracts were separated using online chromatography on a capillary PGC column with a chloroform gradient. By exploiting the retention properties of hydrophobic and polar analytes on PGC, baseline separation of LLOs with minor changes in oligosaccharide structure and polyisoprene chain length was obtained. This method is capable of analyzing low levels of LLOs (from approximately [10.sup.6] bacterial cells) and distinguishing the LLOs that differ by as little as one monosaccharide or polyisoprene unit. Furthermore, we have demonstrated for the first time that oligosaccharides of the C. jejuni Pgl pathway are assembled on different polyisoprenes, e. g. [C.sub.45], [C.sub.60], and apparent hydroxylated forms, in addition to those previously reported (i.e., [C.sub.50] and [C.sub.55]). The hydroxylated forms of the LLOs are believed to be an intermediate in the degradation of accumulated LLOs for polyisoprene carrier recycling. 10.1021/ac9013622

Multiple bacterial species often coexist as communities, and compete for environmental resources. Here, we describe how an opportunistic pathogen, Pseudomonas aeruginosa, uses extracellular products to interact with the nosocomial pathogen Staphylococcus epidermidis. S. epidermidis biofilms and planktonic cultures were challenged with P. aeruginosa supernatant cultures overnight. Results indicated that quorum-sensing-controlled factors from P. aeruginosa supernatant inhibited S. epidermidis growth in planktonic cultures. We also found that P. aeruginosa extracellular products, mainly polysaccharides, disrupted established S. epidermidis biofilms. Cellulase-treated P. aeruginosa supernatant, and supematant from pelA, pslFand pelApslBCD mutants, which are deficient in polysaccharide biosynthesis, diminished the disruption of S. epidermidis biofilms. In contrast, S. epidermidis supernatant in overnight cultures had no effect on established P. aeruginosa biofilms and planktonic growth. These findings reveal that P. aeruginosa extracellular products are important microbial competition factors that overcome competition with S. epidermidis, and the results may provide clues for the development of a novel strategy for controlling S. epidermidis biofilms.

Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescenfly tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-[alpha]-1,5-L-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-[alpha]-1,5-L-arabinan as the preferred substrate, the enzyme exhibited an apparent [K.sub.m] of 2.0 mg [ml.sub.-1] and [V.sub.max] of 0.25 mmol [min.sup.-1] [mg.sup.-1] at pH 7.0 and 50[degrees]C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a [[sigma].sup.A]-dependent and a [[sigma].sup.H]-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.

Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with online LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LFF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans GO, G1, G1' and G2.

The effect of polysaccharide charge density on protein/polysaccharide complexation in solution is studied to understand the effect of polysaccharide charge density on complex adsorption of the air/water interface. It is observed that polysaccharide charge density is a major determinant in the solubility of the protein/polysaccharide complexes.

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantifies of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.

Cyclic [beta]-1,2-glucans (C[beta]G) are osmolyte homopolysaccharides with a cyclic [beta]-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the C[beta]G synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked [beta]-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a [beta]-1,2-glucan phosphorylase present at the C[beta]G synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the [beta]-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this 'lengthcontrolling' phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides. cyclic [beta]-1,2-glucan | phosphorylase | size control

Calcium absorption efficiency and bone mineral mass are increased in adolescents who regularly consume inulin-type fructans (ITF). The mechanism of action in increasing absorption is unknown but may be related to increased colonic calcium absorption. We conducted a study in young adults designed to evaluate these mechanisms with a kinetic technique using [sup.42]Ca orally and [sup.46]Ca dosed i.v. Those who responded to 8 wk of supplementation with 8 g of a mixed short and long degree of polymerization ITF by increasing their calcium absorption had kinetic measurements analyzed to evaluate the time course of absorption. The area under the curve of the oral tracer in the blood during the 26 h after dosing was calculated and the time dependence of increased absorption determined. Eight young adults (of 13 studied), with mean calcium intake ~900 mg/d, responded to the ITF with an increased calcium absorption of at least 3%. In responders, absorption increased from 22.7 [+ or -] 11.3% to 31.0 [+ or -] 15.3%. Colonic absorption, defined as absorption that occurred >7 h after oral dosing, represented 69.6 [+ or -] 18.6% of the increase, or 49 [+ or -] 28 mg/d. These findings suggest that, in those who respond to ITF, its effects on calcium absorption occur principally in the colon. This benefit to ITF may be especially important when absorption in the small intestine is impaired for anatomic or physiological reasons.

A compact biosensor for a label-free, rapid (

Endemic and epidemic shigeliosis, an acute invasive disease of the lower intestines, afflicts millions of people worldwide with an estimated one million fatalities per annum at a low infectious dose. Our approach to vaccine development against Shigella is based on the hypothesis that serum IgG antibodies to the O-specific polysaccharide (O-SP) domains of the LPS of these organisms confer protection to infection. The synthetic oligosaccharides corresponding to the tetrasaccharide repeating unit of the O-SP of Shigella dysenteriae type 1 covalently linked to human serum albumin elicited O-SP-specific IgG in mice. The antibody levels were a function of both the saccharide chain length and their loading on the protein. These synthetic saccharide conjugates elicited significantly higher levels of IgG anti O-SP than conjugates prepared with the O-SP from the bacteria. Here, we evaluated the influence of the nonreducing terminal monosaccharide on the serum antibody response. To this end, we prepared synthetic oligosaccharides comprising hexa- to tridecasaccharide fragments of the native O-SP, having one of the four monosaccharide residues that constitute the repeating unit at their termini and bound them to BSA by a single-point attachment. The conjugates contained an average of 19 saccharide chains per BSA. The synthetic oligosaccharides inhibited the binding of serum raised against whole bacteria to its LPS to a similar extent but lower than the native O-SP. The highest anti-LPS levels were elicited by conjugates having N-acetyiglucosamine (10-met) or galactose residues (7- and 11-mers) at their nonreducing termini. vaccine | glycoconjugate | oligosaccharide synthesis | bioconjugation | dysentery

Lipopolysaccharide (LPS) is recognized as an inducer of the inflammatory response associated with gram-negative sepsis and systemic inflammatory response syndrome. LPS induction proceeds through Toll-like receptor (TLR) in immune cells and intestinal epithelial cells (IEC). This report presents the first identification of Bc110 (B-cell CLL/lymphoma 10) as a mediator of the LPS-induced activation of IL-8 in human IEC. Be110 is a caspase-recruitment domain-containing protein, associated with constitutive activation of NF-[kappa]B in MALT (mucosa-associated lymphoid tissue) lymphomas. The normal human IEC line NCM460, normal primary human colonocytes, and ex vivo human colonic tissue were exposed to 10 ng/ml of LPS for 2-6 h. Effects on Bc110, phospho-I[kappa]B[alpha], NF-[kappa]B, and IL-8 were determined by Western blot, ELISA, immunohistochemistry, and confocal microscopy. Effects of Bc110 silencing by small-interfering RNA (siRNA), TLR4 blocking antibody, TLR4 silencing by siRNA, and an IL-1 receptor-associated kinase (IRAK)-1/4 inhibitor on LPS-induced activation were examined. Following Bc110 silencing, LPS-induced increases in NF-[kappa]B, I[kappa]B[alpha], and IL-8 were significantly reduced (P < 0.001). Increasing concentrations of LPS were associated with higher concentrations of Bc110 protein when quantified by ELISA, and the association between LPS exposure and increased Bc110 was also demonstrated by Western blot, immunohistochemistry, and confocal microscopy. Exposure to TLR4 antibody, TLR4 siRNA, or an IRAK-1/4 inhibitor eliminated the LPS-induced increases in Bc100, NF-[kappa]B, and IL-8. Identification of Bc110 as a mediator of LPS-induced activation of NF-[kappa]B and IL-8 in normal human IEC provides new insight into mechanisms of epithelial inflammation and new opportunities for therapeutic intervention. lipopolysaccharide; inflammation; colonocytes; Toll-like receptor 4

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an [alpha]-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing [alpha]-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-de+pendent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing [alpha]-mannosidase-like protein and Mns1 orthologs, respectively, each of which had [alpha]-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD. protein folding | protists | Trichomonas | Entamoeba

Human hyaluronidase-1 is implicated in cancer proliferation, angiogenesis and inflammatory diseases. Its expression is up regulated in advanced stages of bladder cancer, whereas the expression of the alternative splice variants is down regulated.

The binding of a 12-residue linear peptide, YW 12 rich in aromatic and aliphatic amino acid residues, to lipopolysaccharide (LPS) renders it a well-folded structure. The structural features of YW12 obtained from spectroscopic and fluorescence studies have indicated that interactions only with negative lipid micelles would be useful in the development of nontoxic antisepsis compounds.

A synthetic approach is developed for providing easy access to a wide range of lipid A's by using a common disaccharide building block functionalized with a versatile set of protecting groups. The results have shown that cytokine secretion induced by lipopolysaccharides (LPS) and lipid A is complex, which can be utilized for the development of immuno-modulating therapies.

Synthetic 3-O-methylmannose containing polysaccharide (sMMPS) were designed and synthesized. The procedure involves five glycosidations and produces sMMP-8-, 12-, and 16mers and it was found that the synthetic and natural MMPs exhibit almost similar properties.

Synthetic 6-O-methyl-D-glucose containing polysaccharides (sMGPs) were designed and synthesized from [alpha], [beta] and [gamma]-cyclodextrins (CDs). Results show that the amount of Mukaiyama acid required in the sMGP series increased with an increase of substrate size.

A study presents various cyclic voltammetric (CV) investigations and characterization of the properties of microdynamics in polysaccharide hydrogels and methyl cellulose (MC), coated on glassy carbon (GC) electrodes. The analysis of the various hydrophobic interactions show that the CV method is extremely sensitive and simple and hence provides means to probe the electron-transfer at the MC-GC interface that can be used in the fabrication of new biosensors.

The development of fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the lipopolysaccharide (LPS) -binding protein CD14 is presented. It is shown that an LPS-binding and neutralizing domain of the LPS receptor CD14 constitutes a highly sensitive and specific recognition element for a fluorescence-based LPS sensor.

The sequence analysis of a chromosomal region inside the bce gene cluster from the mucoid clinical isolate Burkholderia cepacia IST408 is presented. The size of the biofilm produced in vitro by bceD and bceF mutant strains is found to be smaller than the size of the biofilm formed by the parental strain, thus showing that BceD and BceF play a vital role in the establishment of biofilms of maximal size.

An extensive molecular modeling study of the [beta]CD-[alpha]HL and selected mutant complexes to gain insights into the [beta]CD-[alpha]HL interaction mechanisms and to predict possible structures and energetics of the complexes, is presented. Results indicated that stability of the direct protein-adapter hydrogen bonds monitored in the course of simulation correlated well with the stability of the complex.