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The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFGsh) showed [greater than or equal to] 57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLMsh genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: --3-[alpha]-LFucNAc-3-(2-NAc-4-N-Asp-2,4, 6-trideoxy-[beta]-D-Glc)-4-[alpha]-D-GicNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.

Streptococcus pneumoniae (the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serolype. The cps loci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. The cps loci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely related cps loci within a subcluster were compared to the known CPS structures to relate gene content to structure. The Streptococcus oralis and Streptococcus mitis polysaccharide biosynthetic loci clustered within the pneumococcal cps loci and were in a subcluster that also included the cps locus of pneumococcal serotype 21, whereas the Streptococcus agalactiae cps loci formed a single cluster that was not closely related to any of the pneumococcal cps clusters.

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan lipoarabinomannan (LAM). This study shows by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, CgLAM, characterized by single t-Araf residues linked to the [alpha](1 [right arrow] 6)-linked mannan backbone. MALDI-TOF MS showed an average molecular mass of 13 800-15 400 Da for Cg-LAM. The biosynthetic origin of Araf residues found in the extracytoplasmic arabinan domain of AG and LAM is well known to be provided by decaprenyl-monophosphoryl-D-arabinose (DPA). However, the characterization of LAM in a C. glutamicum : : ubiA mutant devoid of prenyltransferase activity and devoid of DPA-dependent arabinan deposition into AG revealed partial formation of LAM, albeit with a slightly altered molecular mass. These data suggest that in addition to DPA utilization as an Araf donor, alternative pathways exist in Corynebacterianeae for Araf delivery, possibly via an unknown sugar nucleotide.

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and three L-glycero-D-manno-heptose residues, galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and 044 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1% the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.

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.

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.

B-1a cells reside in both the peritoneal cavity and the spleen. LPS stimulates splenic B-1a to differentiate to plasma cells producing natural IgM specific for microbial and self antigens. However, there are conflicting views as to whether the B-1a cells divide before this differentiation occurs, and hence how the resident B-1a population is maintained in the spleen. Studies here resolve this dispute in favor of both sides: we show that (some or all) B-1a cells resident in the spleen respond to LPS by differentiating to plasma cells immediately, without dividing; however, we also show that additional B-1a cells immigrate into the spleen after LPS stimulation and divide at least once before differentiating. Importantly, the studies we presently describe reveal the complex cell migration and differentiation events that collectively underlie the rapid production of natural antibodies in response to in vivo LPS stimulation. Thus, the studies present a different view of the roles that B-1a cells play in the early phases of the innate immune response. B-1 | CD138 | CD5 | lipopolysaccharide | plasma cells

A new type of hybrid gel matrix, which is prepared by the sol-gel process of tetrakis (2-hydroxyethyl) orthosilicates (THEOS), a completely water-soluble silica precursor, is developed and used for the construction of the amperometric hydrogen peroxide biosensor. The fabricated hydrogen peroxide biosensor has the characteristics of good analytical performance, reproducibility, and storage stability under the optimized experimental conditions.

Capillary eletrophoresis is used to show that Gne catalyze the interconversion of UDP-GlcNAc/GalNAc and UDP-Glc/Gal almost equally well. The comparison of kinetic parameters of Gne from Escherichia coli O86:B7 to those of other characterized UDP-GlcNAc/Glc 4-epimerases suggests that it has relaxed specificity toward the four substrates, the first characterized enzyme to have this activity in the O-antigen biosynthesis.

Vibrio vulnificus causes both food-borne disease and wound infections and most V. vulnificus strains express capsular polysaccharide (CPS), which is required for the virulence of this organism. The wzb gene, which encodes a phosphotase required for CPS expression is proposed to be involved in the switch from opaque {Op, [CPS.sup.+]} colony morphology to a translucent {Tr, [CPS.sup.-]} colony morphology.

NMR spectroscopy of [super 15]N-labeled human [beta]-defensin 2 (HBD2) is used to map the binding sites for two chemokine/glycosaminoglycan (GAG) model compounds, a heparin/heparan sulfate (HS) pentasaccharide (fondaparinux sodium; FX) and enzymatically prepared dermatan sulfate (DS) hexasaccharide (DSdp6). The formation of FX-bridged HBD2 dimers exhibited features of a cooperative binding mechanism while the complex with DSdp6 is found to be mostly monomeric.

A new method is developed for automated glycan synthesis that has used the enzymatic approach and a dendrimer as an ideal support within the chemical process. The protocol has allowed for the fully automated enzymatic synthesis of silyl Lewis X tetrasaccharide derivatives from a simple N-acetyl-D-glucosamine linked to an aminooxy-functionalized G7 dendrimer.

A new modified polysaccharide (Cur-oeg) is designed to form a loose macromolecular complex with a conjugated polymer (CP) that has allowed structural changes in response to external stimuli. The induced thermoresponsiveness is associated with structural rearrangement of the helical conformation of cationic polythiophene (PT1) in the complex and the film color is varied when it is exposed to the vapor of water or methanol, resulting from the structural change of PT1 occurring even in the film state.

Gal:3-O-sulfotransferase-2 (Gal3ST-2) was depleted via siRNA and exogenous Lewis-x trisaccharide 3'-sulfate sodium salt was added in human SMMC7721 hepatoma cells to study the role of Gal3ST-2 and its product 3'-sulfo-[Le.sup.x]. The results revealed the significant role Gal3ST-2 and its product, 3'-sulfo-Lex, towards the regulation of integrin subunit [alpha]V and hence might be associated with cancer cell regulation.

A facile stereoselective total synthesis of cleistenolide from the natural chiral template D-arabinose is achieved by using key steps, including Wittig olefination, selective 1,3-trans-acetal formation and modified Yamaguchi esterification.

Glycolipids are designed and synthesized as simple models of the terminal portion of the mycolyl-arabinogalactan (mAG) in order to probe the flexible scaffold hypothesis. The rotamer populations about the C-4-C-5 bond for the [beta]-linked ring in disaccharide have changed upon acylation at O-5.

Various studies are conducted to determine the way in which different proteins get together to form various lipopolysaccharide (LPS) assemblies in Escherichia coli. The characteristics of the transenvelope complex formed by these proteins are also analyzed.

The application of the various mixtures and assemblies of proteins and polysaccharides in the formation of aragonite complexes is determined. The use of the nacre protein fragment templates for the purpose is also analyzed.

The liquid chromatography and mass spectrometry (LC-MS) was used to compare the compositions of affinity-purified heparin and heparan sulfate (HS) octasaccharides with anticoagulant activities versus library octasaccharides. The method confirmed the isomerically enriched composition of and biological activity of HS.

The catalytic properties of the spnH, spnI and spnK gene products are studied and their roles in the permethylation process of spinosyn A are validated in order to verify the functions of the rhamnose methyltransferases and also their coordination to achieve the desired level of methylation of rhamnose. The studies have shown that the completion of rhamnose permethylation is achieved by the proper control of the expression levels of the methyltransferase genes involved.

The helical structure and conformational analysis of xylose-DNA on the basis of 35 ns molecular dynamics (MD) simulations of a 29-base-pair DNA duplex is presented. The obtained results demonstrated the chiral orthogonality of the ribose and xylose based episomes and the stability and compactness of information storage of the ribose based natural DNA.

The analyses of the dynamic motions of the receptor binding domains (RBDs) and interaction energy profiles with individual glycan residues have helped in identifying residue positions in the RBD that present distinct profiles with the receptor analogues. Glycan binding has constrained the conformational space sampling by the helagglutinins (HA) and electrostatic steering has played a key role in glycan binding specificity.

The synthesis of 2,6-sialylated tridecasaccharide associated with the Fc fragment of intravenous immunoglobulin is described.

Glycan microarray profiling, NMR spectroscopy and mutagenesis have shown that one of the two oligomannose bidning sites of Microcystis viridis lectin (MVL) has catalyzed the cleavage of chitin fragments to GlcNAc in order to determine the mode of MVL binding to and cleavage of chitotriose. The studies have shown the dual catalytic activity and carbohydrate recognition for discrete oligosaccharides at the same carbohydrate-binding site in a lectin.

A new 'in situ' enzymatic elongation methodology that facilitated for the first time the isolation and structural characterization of a catalytically competent covalent glycosyl-enzyme intermediate with human pancreatic [alpha]-amylase is presented. The scope of the elongation approach to probe the active sites and catalytic mechanisms of [alpha]-amylases through the preliminary experiments with porcine pancreatic [alpha]-amylase is demonstrated.

A simple, mild, and chemoselective strategy is developed for the conjugation of biologically active molecules to the surface of dextran-based microparticles. The functionalization of dextran-based microparticles with a fluorophore as well as a cell-penetrating peptide sequence, which facilitated the delivery of cargo to nonphagocytic cells leading to a 60-fold increase in the expression of a reporter gene in presence of plasmid DNA-loaded particles, is also demonstrated.

A series of mechanistic biochemical studies was conducted with purified native PglH, a heptasaccharide assembly enzymes to understand the mechanism whereby the reactions cease after the addtion of just three GalNAc residues. The results provided important mechanistic insights into the function of the glycosyl transfer polymerase that is related to the virulence of human gut mucosal pathogen Campylobacter jejuni.

A study is conducted to examine the cation-exchange properties of Pseudomonas aeruginosa PA01 and PA01 wbpL cells grown in the presence or absence of magnesium and subsequently exposed to sodium, calcium, or lanthanum. The main metal-binding regions of PA01 are found to be located in the core section of the lipopolysaccharide (LPS) and hence suggested that the selection of O-antigen traits does not affect the metal-binding ability of the cell.

Escherichia coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster was generated to study the role of the O antigen in persistence and colonization in the animal host. The data results suggested that the O polysaccharide side chain of Escherichia coli O157:H7 lipopolysaccharide (LPS) is a required factor for efficient bovine intestinal colonization.