Unusual polysaccharide rheology of aqueous dispersions of soft phytoglycogen nanoparticles.

Phytoglycogen is a natural polysaccharide produced in the form of dense, 35 nm diameter nanoparticles by some varieties of plants such as sweet corn. The highly-branched, dendrimeric structure of phytoglycogen leads to interesting and useful properties such as softness and deformability of the particles, and a strong interaction with water. These properties make the particles ideal for use as unique additives in personal care, nutrition and biomedical formulations. In the present study, we describe rheology measurements of aqueous dispersions of phytoglycogen nanoparticles. The viscosity of the dispersions remained Newtonian up to large concentrations (∼20% w/w). For higher concentrations, the zero-shear viscosity increased dramatically, reaching values that exceeded that of the water solvent by six orders of magnitude at a concentration of 30% w/w and were well described by the Vogel-Fulcher-Tammann relation of glassy dynamics. The very large values of the zero-shear viscosity are coupled with significant deformation of the soft nanoparticles. We quantified the softness of the particles by performing osmotic pressure measurements on concentrated dispersions, obtaining a value of 15 kPa for the compressional modulus. For the most concentrated samples, we observed flow at stresses less than the apparent yield stress value determined by fitting the high strain rate data to the Herschel-Bulkley model. This behavior, similar to that of star polymer glasses, suggests the possibility of a hairy colloid particle geometry. Remarkably, phytoglycogen nanoparticles dispersed in water provide a very simple experimental realization of glass-forming dispersions of soft colloidal particles that can be used to validate theoretical models in detail.

Structure and Hydration of Highly-Branched, Monodisperse Phytoglycogen Nanoparticles.

Phytoglycogen is a naturally occurring polysaccharide nanoparticle made up of extensively branched glucose monomers. It has a number of unusual and advantageous properties, such as high water retention, low viscosity, and high stability in water, which make this biomaterial a promising candidate for a wide variety of applications. In this study, we have characterized the structure and hydration of aqueous dispersions of phytoglycogen nanoparticles using neutron scattering. Small angle neutron scattering results suggest that the phytoglycogen nanoparticles behave similar to hard sphere colloids and are hydrated by a large number of water molecules (each nanoparticle contains between 250% and 285% of its mass in water). This suggests that phytoglycogen is an ideal sample in which to study the dynamics of hydration water. To this end, we used quasielastic neutron scattering (QENS) to provide an independent and consistent measure of the hydration number, and to estimate the retardation factor (or degree of water slow-down) for hydration water translational motions. These data demonstrate a length-scale dependence in the measured retardation factors that clarifies the origin of discrepancies between retardation factor values reported for hydration water using different experimental techniques. The present approach can be generalized to other systems containing nanoconfined water.

Differential high-affinity interaction of dectin-1 with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and side-chain branching.

Glucans are structurally diverse fungal biopolymers that stimulate innate immunity and are fungal pathogen-associated molecular patterns. Dectin-1 is a C-type lectin-like pattern recognition receptor that binds glucans and induces innate immune responses to fungal pathogens. We examined the effect of glucan structure on recognition and binding by murine recombinant Dectin-1 with a library of natural product and synthetic (1-->3)-beta/(1-->6)-beta-glucans as well as nonglucan polymers. Dectin-1 is highly specific for glucans with a pure (1-->3)-beta-linked backbone structure. Although Dectin-1 is highly specific for (1-->3)-beta-d-glucans, it does not recognize all glucans equally. Dectin-1 differentially interacted with (1-->3)-beta-d-glucans over a very wide range of binding affinities (2.6 mM-2.2 pM). One of the most striking observations that emerged from this study was the remarkable high-affinity interaction of Dectin-1 with certain glucans (2.2 pM). These data also demonstrated that synthetic glucan ligands interact with Dectin-1 and that binding affinity increased in synthetic glucans containing a single glucose side-chain branch. We also observed differential recognition of glucans derived from saprophytes and pathogens. We found that glucan derived from a saprophytic yeast was recognized with higher affinity than glucan derived from the pathogen Candida albicans. Structural analysis demonstrated that glucan backbone chain length and (1-->6)-beta side-chain branching strongly influenced Dectin-1 binding affinity. These data demonstrate: 1) the specificity of Dectin-1 for glucans; 2) that Dectin-1 differentiates between glucan ligands based on structural determinants; and 3) that Dectin-1 can recognize and interact with both natural product and synthetic glucan ligands.

Effect of cations on the structure of bilayers formed by lipopolysaccharides isolated from Pseudomonas aeruginosa PAO1.

The asymmetric outer membrane of Gram-negative bacteria contains lipopolysaccharides (LPSs) which contribute significantly to the bacterium's surface properties and play a crucial role in regulating membrane permeability. We report on neutron diffraction studies performed on aligned, self-assembled bilayers of Na-, Ca-, and Mg-salt forms of LPS isolated from Pseudomonas aeruginosa PAO1. From the one-dimensional neutron scattering length density profiles we find that water penetrates Ca2+-LPS bilayers to a lesser extent than either Na+- or Mg2+-LPS bilayers. This differential water penetration could have implications as to how small molecules permeate the outer membrane of Gram-negative bacteria and, possibly, how nonlamellar phases are formed.

Cell-surface alpha-glucan in Campylobacter jejuni 81-176.

Campylobacter jejuni infection is a main source of severe gastroenteritis-related illnesses in humans and there is also evidence that it may be linked to neurological disorders. C. jejuni 81-176 is a virulent strain that has become the global model in the study of mechanisms and pathogenesis of C. jejuni infection. For this reason, we were engaged in studying the fine structures of cell-surface carbohydrate antigens of C. jejuni 81-176, namely, the capsule polysaccharide (CPS) and lipooligosaccharide (LOS). Serologically, C. jejuni 81-176 has been classified as belonging to serogroups HS23 and HS36, and indeed previous studies have shown that the LOS and CPS structures possess components similar to those expressed by serostrains HS23 and HS36. Here, we describe that in addition to the LOS and CPS, this strain also produced an independent cell-surface (1-->4)-alpha-glucan capsule.

A novel Helicobacter pylori cell-surface polysaccharide.

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.

Mutagenicity of the oral carcinogen 4-nitroquinoline-1-oxide in cultured BigBlue rat tongue epithelial cells and fibroblasts.

Environmental carcinogen exposures contribute to the development of oral cancer and improved test systems for the analysis of such carcinogens are needed. We have previously isolated and characterized an epithelial cell line from the tongue of a BigBlue rat. Now, we have established an immortalized fibroblast cell line from the same organ. We exposed these cells to 4-nitroquinoline-1-oxide (NQO), a well-known experimental oral carcinogen in the rat and other species, and measured its cytotoxic and genotoxic (cII transgene mutagenesis) effects. Both cell lines were very sensitive to NQO toxicity and showed dose-dependent mutant frequency responses. At the highest NQO dose tested, 70 ng/ml, the mutant frequency was elevated more than eight-fold above background for the epithelial cells and more than 25-fold for the fibroblast cells. We examined cellular parameters which could affect glutathione-dependent detoxication of mutagens. Glutathione (GSH) contents of the two cell lines were similar. Glutathione transferase (GST) activities were measured with several substrates and were generally higher in the epithelial cells. Although multiple biochemical and biological characteristics of individual cell lines are likely to determine responses to mutagens, the greater sensitivity of the fibroblast cells to NQO mutagenicity is in accord with the lower GST activity and the lower DNA content of these cells. These new cell lines are suitable for in vitro testing of chemicals as possible oral mutagens and for studies of their biochemical mechanisms of action.

Oil binding capacities of triacylglycerol crystalline nanoplatelets: nanoscale models of tristearin solids in liquid triolein.

Polycrystalline particles composed of triacylglycerol (TAG) molecules, and their networks, in anhydrous TAG oils find extensive use as edible oils in the food industry. Although modelling studies of TAG systems, have been carried out, none have attempted to address a problem of central concern to food science and technology: the "oil binding capacity" of a system of such edible oils. Crystalline nanoparticles (CNPs) have recently been identified as the fundamental components of solid fats in oils. Oil binding capacity is an important concept regarding the ability of fats particles to retain oil, and the ability of these CNPs to bind oil is important in designing healthy foods. We have carried out atomic scale molecular dynamics computer simulations to understand the behavior of a triacylglycerol oil (triolein) in nanoscale confinements between tristearin CNPs. We define a nanoscale oil binding capacity function by utilizing the average oil number density, 〈Φ(d)〉, between two CNPs as a function of their separation, d. We modelled pure tristearin CNPs as well as tristearin CNPs in which the surfaces are covered with an interface comprising soft permanent coatings. Their surfaces are "hard" and "soft" respectively. We found that for a pair of hard-surface tristearin CNPs a distance d apart, (i) triolein exhibits number density, and therefore density, oscillations as a function of d, and (ii) the average number density between two such CNPs decreases as d decreases, viz. the oil binding capacity is lowered. When a soft layer of oil covers the CNP surfaces, we found that the oscillations are smeared out and that the average number density between the two CNPs remained approximately constant as d decreased indicating a high oil binding capacity. Our results might have identified important nanoscale aspects to aid in healthy food design.

Nanoscale characteristics of triacylglycerol oils: phase separation and binding energies of two-component oils to crystalline nanoplatelets.

Fats are elastoplastic materials with a defined yield stress and flow behavior and the plasticity of a fat is central to its functionality. This plasticity is given by a complex tribological interplay between a crystalline phase structured as crystalline nanoplatelets (CNPs) and nanoplatelet aggregates and the liquid oil phase. Oil can be trapped within microscopic pores within the fat crystal network by capillary action, but it is believed that a significant amount of oil can be trapped by adsorption onto crystalline surfaces. This, however, remains to be proven. Further, the structural basis for the solid-liquid interaction remains a mystery. In this work, we demonstrate that the triglyceride liquid structure plays a key role in oil binding and that this binding could potentially be modulated by judicious engineering of liquid triglyceride structure. The enhancement of oil binding is central to many current developments in this area since an improvement in the health characteristics of fat and fat-structured food products entails a reduction in the amount of crystalline triacylglycerols (TAGs) and a relative increase in the amount of liquid TAGs. Excessive amounts of unbound, free oil, will lead to losses in functionality of this important food component. Engineering fats for enhanced oil binding capacity is thus central to the design of more healthy food products. To begin to address this, we modelled the interaction of triacylglycerol oils, triolein (OOO), 1,2-olein elaidin (OOE) and 1,2-elaidin olein (EEO) with a model crystalline nanoplatelet composed of tristearin in an undefined polymorphic form. The surface of the CNP in contact with the oil was assumed to be planar. We considered pure OOO and mixtures of OOO + OOE and OOO + EEO with 80% OOO. The last two cases were taken as approximations to high oleic sunflower oil (HOSO). The intent was to investigate whether phase separation on a nanoscale took place. We defined an "oil binding capacity" parameter, B(Q,Q'), relating a state Q to a reference state Q'. We used atomic scale molecular dynamics in the NVT ensemble and computed averages over 1-5 ns. We found that the probability of the OOE phase separating into a layer on the surface of the CNP compared to being retained randomly in an OOO + OOE mix were approximately equal. However, we found that it was probable that the EEO component of an OOO + EEO mix would phase separate and coat the surface of the CNP. These results suggest a mechanism whereby many-component oils undergo phase separation on a nanoscale so as to create a transition oil region between the surface of the CNP and the bulk major oil component (OOO in the case considered here) so as to create the appropriate oil binding capacity for the use to which it is put.

Calcium ions induce collapse of charged O-side chains of lipopolysaccharides from Pseudomonas aeruginosa.

Lipopolysaccharide (LPS) monolayers deposited on planar, hydrophobic substrates were used as a defined model of outer membranes of Pseudomonas aeruginosa strain dps 89. To investigate the influence of ions on the (out-of-plane) monolayer structure, we measured specular X-ray reflectivity at high energy (22 keV) to ensure transmission through water. Electron density profiles were reconstructed from the reflectivity curves, and they indicate that the presence of Ca(2+) ions induces a significant change in the conformation of the charged polysaccharide head groups (O-side chains). Monte Carlo simulations based on a minimal computer model of LPS molecules allow for the modelling of 100 or more molecules over 10(-3) s and theoretically explained the tendency found by experiments.

The structural basis for the serospecificity of Actinobacillus suis serogroup O:2.

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1-->6)-beta-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: -->3)-beta-D-Galp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc-(1-->. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.

Mutation of waaC, encoding heptosyltransferase I in Campylobacter jejuni 81-176, affects the structure of both lipooligosaccharide and capsular carbohydrate.

Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first L-glycero-D-manno-heptose residue to 3-deoxy-D-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

Microenvironmental influences on mutagenesis in mammary epithelial cells.

Tumor progression may be viewed as an evolutionary process at the cellular level. Because blood supply to solid tumors is inadequate, the cancer cells face a hostile microenvironment characterized by hypoxia or anoxia, acidic extracellular pH and nutrient deficiencies. It has been proposed that these factors result in increased levels of spontaneous mutagenesis and thereby contribute to tumor progression. We have examined spontaneous mutagenesis in vitro and in vivo, using previously characterized cell lines (mammary epithelial cells [ME] and mammary fibroblast cells [MFib]) from the mammary gland of the BigBluetrade mark rat, carrying a transgene construct suitable for the detection of mutations. Cells were exposed in vitro to control conditions, low pH, or to glucose deprivation, under normoxic or hypoxic culture conditions, and were also grown as xenografted tumors in immune-deficient mice. We examined cell survival and mutant frequency/spectrum at the cII locus. Significant increases in mutant frequency were observed in ME cells exposed to hypoxia alone or in combination with no glucose; the latter condition also resulted in reduced clonogenic survival. Cells grown as xenografts and then recovered and expanded in culture also had elevated frequencies of spontaneous mutations. We observed a shift in the spontaneous mutation spectrum between the ME cells and the MET cells (cultured in vitro or isolated from mouse xenograft tumors). These results support the concept that the tumor microenvironment contributes to tumor progression by enhancing spontaneous mutagenesis, that different cell types from the same organ can respond differently to these stresses and that differences in microenvironment may influence the types of mutations that arise.