Antifracture, Antibacterial, and Anti-inflammatory Hydrogels Consisting of Silver-Embedded Curdlan Nanofibrils.

The bacterial exopolysaccharide Curdlan has a unique collagen-like triple helical structure and immune-modulation activities. Although there have been several types of Curdlan gels reported for antibacterial or wound healing purposes, none of them exhibit favorable mechanical properties for clinically applicable wound healing materials. Herein, we present a two-step approach for preparing Ag-embedded Curdlan hydrogels that are highly soft but are very stretchable compared with common polysaccharide-based hydrogels. Ag ions were first reduced in a diluted Curdlan solution to form AgNP-decorated triple helices. Then, the aqueous solution consisting of Curdlan/Ag nanoparticles was mixed with a dimethyl sulfoxide solution consisting of a high concentration of Curdlan. This mixing triggered the conformation transformation of Curdlan random coils into triple helices, and then the helices were further packed into semicrystalline nanofibrils of ∼20 nm in diameter. Due to the presence of semicrystalline fibrils, this novel Curdlan hydrogel exhibits a fracture strain of ∼350% and fracture stress of ∼0.2 MPa at a water content of ∼97%. This nanofibril hydrogel supported the attachment, spreading, and growth of fibroblasts and effectively inhibited the growth of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Moreover, the hydrogels downregulated NO production and proinflammatory gene expression levels in lipopolysaccharide (LPS)-stimulated macrophages but did not change the anti-inflammatory gene expression levels in IL-4-stimulated macrophages. In an animal study, these hydrogels accelerated wound healing in a bacteria-infected mice skin wound model. These results validate the further development of Curdlan/AgNPs nanofibril hydrogels in clinical wound management.

Unraveling the Structural Modifications in Lignin of Arundo donax Linn. during Acid-Enhanced Ionic Liquid Pretreatment.

Solid acid-enhanced ionic liquid (IL) pretreatment is of paramount importance for boosting the yield of sugars from biomass cost-effectively and environmentally friendly. To unravel the chemical and supramolecular structural changes of lignin after pretreatment, IL-acid lignin (ILAL) and subsequent residual cellulolytic enzyme lignin (RCEL) were isolated from Arundo donax Linn. The structural features were compared with those of the corresponding milled wood lignin (MWL). Results indicated that the pretreatment caused loss of ß-O-4', ß-ß', ß-1' linkages and formation of condensed structures in lignin. A preferential breakdown of G-type lignin may have occurred, evidenced by an increased S/G ratio revealed by 2D HSQC NMR analysis. It was determined that the depolymerization of ß-O-4' linkage, lignin recondensation, and cleavage of ferulate-lignin ether linkages took place. Moreover, a simulation module was first developed to define morphological changes in lignin based on AFM and TEM analyses. Briefly, tree branch like aggregates was destroyed to monodisperse particles.

Micro-CT imaging of denatured chitin by silver to explore honey bee and insect pathologies.

BACKGROUND: Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term "denatured chitin" calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. METHODOLOGY/PRINCIPAL FINDINGS: A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. CONCLUSIONS/SIGNIFICANCE: The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an additional tool for research on CCD.

Stereo-specific inhibition of acetyl- and butyryl-cholinesterases by enantiomers of cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate.

Enantiomers of cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate show stereo-specific inhibition for acetylcholinesterase and butyrylcholinesterase. For both inhibition reaction, (2S,4aR,8aS)-cis,cis-decahydro-2-naphthyl-N-n- butylcarbamate is more potent than (2R,4aS,8aR)-cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate. Optically pure (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthols are resolved by the porcine pancreatic lipase-catalyzed acetylation of decahydro-2-naphthols with vinyl acetate. Absolute configurations and the enantiomeric excess values of (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthols are determined from the (19)F NMR spectra of their Mosher's ester derivatives. We fail to resolve (2S,4aR,8aR)- and (2R,4aS,8aS)-trans,cis-decahydro-2-naphthols from the porcine pancreatic lipase-catalyzed acetylation of decahydro-2-naphthols with vinyl acetate.

Photon transmission study on conformational ordering of iota-carrageenan in CaCl2 solution.

Coil-to-double helix (c-h) and double helix-to-dimer (h-d) phase transitions of iota-carrageenan in CaCl(2) solution upon cooling were studied using photon transmission technique. Photon transmission intensity, I(tr) was monitored against temperature to determine the (c-h) and (h-d) transition temperatures (T(ch) and T(hd)) and activation energies (DeltaE(ch) and DeltaE(hd)). An extra dimer-to-dimer (d-d) transition was also observed during cooling at low temperature region. However, upon heating dimers disappear to double helices by making dimer-to-double helix (d-h) transition. Further heating resulted double helix-to-coil (h-c) transition at high temperature region. T(dh) and T(ch) temperatures and DeltaE(dh) and DeltaE(hc) activation energies were also determined. It was observed that T(hc) and T(ch) temperatures and DeltaE(dh) and DeltaE(hd) activation energies do not effected by carrageenan content. However, T(hd), T(dh) and T(dd) temperatures and DeltaE(ch) and DeltaE(hc) activation energies were found to be strongly correlated to the carrageenan content in the system.

Comparison of kifunensine and 1-deoxymannojirimycin binding to class I and II alpha-mannosidases demonstrates different saccharide distortions in inverting and retaining catalytic mechanisms.

Mannosidases are key enzymes in the eukaryotic N-glycosylation pathway. These enzymes fall into two broad classes (I and II) and are characteristically different in catalytic mechanism, sequence, and structure. Kifunensine is an alkaloid that is a strong inhibitor against class I alpha-mannosidases but is only a weak inhibitor against class II alpha-mannosidases. In this paper, the 1.80 A resolution crystal structure of kifunensine bound to Drosophila melanogaster Golgi alpha-mannosidase II (dGMII) is presented. Kifunensine adopts a (1,4)B boat conformation in the class II dGMII, which contrasts the (1)C(4) chair conformation seen in class I human endoplasmic reticulum alpha1,2 mannosidase (hERMI, PDB ). The observed conformations are higher in conformational energy than the global minimum (4)C(1) conformation, although the conformation in hERMI is closer to the minimum, as supported by an energy calculation. Differing conformations of 1-deoxymannojirimycin were also observed: a (4)C(1) and (1)C(4) conformation in dGMII and hERMI, respectively. Thus, these two alpha-mannosidase classes distort these inhibitors in distinct manners. This is likely indicative of the binding characteristics of the two different catalytic mechanisms of these enzymes.

Membrane disruption and cytotoxicity of hydrophobic N-alkylated imino sugars is independent of the inhibition of protein and lipid glycosylation.

The N-alkyl moiety of N-alkylated imino sugars is crucial for therapeutic activities of these compounds as inhibitors of glycosphingolipid (GSL) biosynthesis and as antivirals. The improved potency afforded by a long N-alkyl moiety is coincident with increased compound-induced cytotoxicity. Therefore, in the present study, we examined the mechanism of this cytotoxicity in detail. Despite N-butyl-deoxynojirimycin and N-butyl-deoxygalactonojirimycin inhibiting the glycosylation of ceramide to glucosylceramide, ceramide levels did not increase in HL60 cells treated with these compounds. Long-chain N-alkylated imino sugars were toxic to cells at concentrations considerably lower than the critical micellar concentrations for these compounds and consequently did not solubilize radioactively labelled cellular proteins and lipids. However, membrane disruption and cell fragmentation did increase in a concentration- and chain-length-dependent manner. These results are consistent with previously proposed interactions between surface-active amphiphiles and protein-containing lipid membranes when drug concentrations are below the critical micellar concentration. Taken together, these results demonstrate that the cellular toxicity of hydrophobic N-alkylated imino sugars is due to cell lysis and cell fragmentation and, most importantly, is not related to the beneficial therapeutic effects of these compounds on protein and in lipid glycosylation. This information will aid in the future development of more selective imino sugar therapeutics for the treatment of human disease.

Retrocyclin, an antiretroviral theta-defensin, is a lectin.

Theta-defensins are circular octadecapeptides that contain an internal tridisulfide ladder. Because retrocyclin-1, an ancestral hominid theta-defensin, can protect human cells in vitro from infection by T- and M-tropic strains of HIV-1, we used surface plasmon resonance techniques to study its binding to glycoproteins and glycolipids implicated in HIV-1 entry. Retrocyclin-1 bound with high affinity to gp120 (K(d), 35.4 nM), CD4 (K(d), 31 nM), and galactosylceramide (K(d), 24.1 nM). Neither a circular form of retrocyclin without its tridisulfide ladder nor its beta-hairpin precursor with these disulfides intact bound gp120 or CD4 effectively. Retrocyclin also bound fetuin, an extensively glycosylated protein, with high affinity, but it did not bind nonglycosylated gp120 or BSA. However, retrocyclin did bind to a neoglycoprotein, BSA, with covalently attached sugar residues. Experiments with glycosidase-treated fetuin, gp120, and CD4 revealed that both O-linked and N-linked sugars were used as binding sites. In a panel of retrocyclin variants, binding to immobilized gp120 and CD4 were highly correlated to each other and to the peptide's ability to protect human PBMCs from infection by HIV-1. Although small, cysteine-rich antimicrobial peptides with lectin-like properties exist in plants, theta-defensins are the first such molecules to be identified in vertebrates. Retrocyclin's ability to recognize and bind carbohydrate-containing surface molecules is integrally related to its ability to protect cells from HIV-1 infection.

Molecular basis of C(2+)-induced gelation in alginates and pectins: the egg-box model revisited.

For many ionic polysaccharides, the ability to form gels in the presence of divalent cations such as calcium is the key to biological functions and technological applications. This is particularly true for alginates and pectins, where the regular occurrence of respectively alpha-L-(1-4)-guluronate residues and alpha-D-galacturonate residues generates ordered templates for polymer chain associations that are involved in physical gels. The molecular basis responsible for the strength and the stereospecificity of calcium interactions for the two polysaccharides were investigated in a previous paper (Braccini; et al. Carbohydr. Res. 1999, 119). In the present work, a novel molecular modeling procedure has been developed; it involves a pairing procedure that evaluates all the possible associations of the ordered polyuronate chains with calcium ions to form dimers. Starting from the stable ordered forms of polygalacturonate and polyguluronate, all possible ways to form Ca(2+)-bridged dimers were computed; the parallel and antiparallel relative arrangements of the chains were also considered. Despite the structural analogy between polyguluronate and polygalacturonate chains, significant differences at the level of chain-chain associations are found. The popular "egg box model" can still be referred to in the case of polyguluronate. However, it cannot be used to describe a pectate junction zone as the unique feature of two consecutive chelation site per repeat, that provides a favorable entropic contribution to the interchain association is not reproduced by this pioneering model. The body of these results corroborates the two-stage process in the mechanism of calcium gelation, where the formation of strongly linked dimer associations is followed by the formation of weak inter-dimer associations mainly governed by electrostatic interactions.

Exploitation of reactivity and selectivity in cellulose functionalization using unconventional media for the design of products showing new superstructures.

A variety of new cellulose solvents was investigated toward their potential as media for the functionalization of the polyglucane. Thus, mixtures of dimethyl sulfoxide (DMSO)/tetrabutylammonium fluoride trihydrate (TBAF), N-methylmorpholine-N-oxide (NMMNO)/DMSO, melts of LiClO(4).3H(2)O, and aqueous solutions of Ni(tren)(OH)(2) [tren = tris(2-aminoethyl)amine] were applied as reaction media. In case of the new solvent, DMSO/TBAF its usefulness for derivatization reactions including the etherification with sodium monochloroacetate and the acylation with vinyl esters of carbonic acids was studied. The structural features of the products were analyzed by means of (1)H NMR spectroscopy (after depolymerization or peresterification), (13)C NMR spectroscopy, and HPLC after complete hydrolytic chain degradation. The results were compared with those obtained from derivatives prepared using the solvent N,N-dimethylacetamide (DMAc)/LiCl and conventional, heterogeneous synthesis. It can be shown that in case of carboxymethylation reactions the reaction medium applied has a drastic influence both on the course of reaction and on the structural features of the products. A highly efficient tool was found to be atomic force microscopy (AFM), showing remarkable differences in the superstructures of the differentially synthesized derivatives.

Single molecule study of xanthan conformation using atomic force microscopy.

Conformations of individual macromolecules of the biopolymer xanthan were investigated using atomic force microscopy (AFM). Xanthan from very dilute solutions (1 ppm) was allowed to adsorb onto freshly cleaved mica and examined using tapping mode AFM under ambient conditions. The secondary structure of xanthan was probed by heating the polymer and gradually cooling, which denatured and renatured the polymer. When salt was present, renatured xanthan formed a double helical structure, consistent with the structure of native xanthan. In pure water, renaturation was not complete as what appeared to be single helical structures were observed. The number-average contour length (L(n)) of the polymer in its single helical state was 1651 nm. In the double helical state, induced by the addition of salt, L(n) decreased to 450 nm (in 0.5 M KCl). The chains also became less rigid as salt was added. The persistence length decreased from 417 nm in pure water to approximately 150 nm in 0.1 or 0.5 M KCl. This indicated a trend toward more flexible molecules when salt was present. Calculations of end-to-end distances based on equilibrium and projected conformations confirmed that the xanthan chain conformation on the mica surface was at equilibrium and was therefore representative of the conformation of xanthan in solution. The single-molecule AFM technique eliminates one common bias of solution techniques, which is the determination of an average signal between aggregates and dissolved molecules. It is thus a useful complement to solution-based methods for determining physical-chemical properties of biopolymers.

Solution conformation of various uridine diphosphoglucose salts as probed by NMR spectroscopy.

The solution conformations of uridine diphosphoglucose (UDP-Glc) under a variety of conditions (solvent, ionic strength, various mono- and divalent cations) have been studied by NMR spectroscopy (1H, 13C, 31P, and 25Mg). In the case of divalent cations (Ca2+, Mg2+, Mn2+) the phosphate oxygens are the preferred coordination sites and analysis of the 25Mg linewidths of solutions with various [Mg2+]/[UDP-Glc] ratios, indicates that the 1:1 Mg2+ UDP-Glc complex is the major species. From 13C relaxation data and hydrodynamic theory, it has been demonstrated that under all conditions UDP-Glc adopts a fairly extended overall shape and that magnesium ions lead to a significant increase in the average length of the UDP-Glc molecule as compared to monovalent cations. Thus, one of the roles of the metal ion in enzymic reactions involving nucleotide sugars may be to preorganize the nucleotide sugar.

Dynamic oscillation measurements of starch networks at temperatures above 100 degrees C.

Small deformation oscillatory studies were performed on wheat flour paste with a starch content of 75.4%. Work focused on temperatures above 100 degrees C in an effort to seek molecular understanding of such high-temperature processes as bakery operations which are characterised by evaporation of water. The moisture content of the sample decreased from about 32% at 100 degrees C to 6.5% at 130 degrees C. Viscoelastic spectra produced a sigmoidal profile with a disproportionate viscous element also seen in the glass transition of semiamorphous synthetic polymers and high sugar/polysaccharide mixtures during cooling. It is argued that the loss of water upon heating reduces the available free volume between neighbouring chain segments, thus generating a high-density thermoplastic melt suspending granule fragments. The configurational rearrangements of the disordered chains contribute mainly to an energy-dissipating process, as observed in the vitrification of cooled high-solids systems. The equation of Williams, Landel, and Ferry was modified with a 'moisture term' in order to describe the temperature function of viscoelasticity.

The phase transformations in starch during gelatinisation: a liquid crystalline approach.

The analogy between starch and a chiral side-chain polymeric liquid crystal is examined in relation to the processes involved during gelatinisation. There are three important parameters for characterisation of the molecular phase behaviour of the amylopectin: the lamellar order parameter (psi), the orientational order parameter of the amylopectin double helices (phi), and the helicity of the sample (h, the helix/coil ratio, a measure of the helix-coil transition of the double helices). The coupling between the double helices and the backbone through the flexible spacers is affected dramatically by the water content and it is this factor which dictates the particular phase adopted by the amylopectin inside the starch granule as a function of temperature. SAXS, WAXS and 13C CP/MAS NMR are used to examine these phenomena in excess water. Furthermore, previous experimental evidence pertaining to the limiting water case is reviewed with respect to this new theoretical framework.

Hydration of pectic polysaccharides.

The hydration and swelling of pectic polysaccharides was examined at different pHs and ionic strengths as a function of osmotic stress. For weakly charged pectic polysaccharides at low concentrations of a monovalent salt (20 mM), the main driving force for swelling originates from a polyelectrolyte effect due to the translational entropy of ions within the film. Swelling is reduced at higher salt concentrations and lower pHs. Polyelectrolyte collapse and minimal swelling is observed for more highly charged pectic polysaccharides. Replacement of the Na(+) counterion with Ca(2+) results in minimal swelling and the formation of network structures even for the weakly charged pectic polysaccharides.

Molecular dynamics simulations of cyclohenicosakis-[(1-->2)-beta-D-gluco-henicosapyranosyl], a cyclic (1-->2)-beta-D-glucan (a 'cyclosophoraose') of DP 21.

We report molecular dynamics simulations of cyclohenicosakis-[(1-->2)-beta-D-gluco-henicosapyranosyl], termed 'cyclosophohenicosamer', a member of a class of cyclic (1-->2)-beta-D-glucans ('cyclosophoraoses'). Our goals were to provide insights into the conformational preferences of these cyclosophoraoses. Simulated annealing and constant-temperature molecular dynamics calculations were performed on the DP 21 cyclosophohenicosamer. The radius of gyration (R(G)) of the molecule and the conformation of glycosidic dihedral angles were used to analyze the result of computational studies. Most glycosidic linkages were concentrated in the lowest-energy region of the phi-psi energy map, and the values of radius of gyration from our simulations were consistent with the reported experimental value. The simulations produced various types of compact and asymmetric conformations within reasonable ranges of the glycosidic linkage conformation and radius of gyration. The results indicate the presence of a high degree of molecular flexibility of cyclosophohenicosamer and suggest the uniqueness of inclusion complexation with other molecules through this molecular flexibility.

Computational studies on carbohydrates: solvation studies on maltose and cyclomaltooligosaccharides (cyclodextrins) using a DFT/ab initio-derived empirical force field, AMB99C.

An empirical force field, denoted AMB99C, has been used to study molecular properties of alpha-(1-->4)-linked carbohydrates in solution. AMB99C was parameterized using structural and energetic parameters from density functional ab initio methodology. In this work we examine the solution behavior of the beta anomer of maltose and cyclohexa-, cyclohepta-, and cyclooctaamyloses (alpha-, beta-, and gamma-cyclodextrins or alpha-, beta-, and gamma-CDs, respectively), as well as of two larger (DP 10, epsilon-CD; DP 21) cyclomaltooligosaccharides, CA10 and CA21. Experimental data used for comparison purposes include X-ray structures, small-angle scattering radius of gyration values, NMR nuclear Overhauser enhancements (NOEs), and proton coupling constants. Molecular dynamics simulations were carried out using explicit water molecules (TIP3P) to establish equilibrium populations of conformations in solution, and these results are compared with other calculated values and a variety of experimental parameters, such as average H-1-H-4' distances between the rings in beta-maltose, and the primary hydroxyl groups' conformational populations. Medium-to-large cyclomaltooligosaccharide molecules were studied to test for glucose ring puckering and stability of kinked and 'flipped' conformations. The results of the solvation studies are in excellent agreement with experimental structural parameters.

The structure of gellan in dilute aqueous solution.

A full assignment of high-field nmr spectra of gellan was obtained in dilute aqueous solution by performing a series of selective one-dimensional nmr experiments. The observed nuclear Overhauser effects (NOEs) cannot be interpreted assuming that each sugar residue is intrinsically rigid and in a chair conformation. In fact, the rhamnose residue gives strong NOE contacts coherent only with an equilibrium involving both a chair as well as a boat (or a hemiboat) conformation. Molecular dynamic calculations performed on a heptamer with a central rhamnose support the above finding, and show a structure based on a very stiff single chain in which it is present a flipping of the rhamnose residue. At low temperatures (5-20 degrees C) in very dilute solutions (0.018 mg/mL) nmr spectra show a splitting of the resonance due to the methyl group of rhamnose residue, thus confirming the presence of a slow equilibrium among different conformers.

N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors.

The possible role of carbohydrate in the interaction of HLA-C with a human inhibitory natural Killer cell Immunoglobulin-like Receptor with two Ig domains, KIR2DL1, was investigated. Transfectants of 721.221 (a class I MHC-negative human B cell line) expressing only HLA-Cw4 or -Cw6 or their respective non-glycosylated mutants (N86Q, S88A) were made. The binding of a KIR2DL1-Ig fusion protein to the non-glycosylated mutant HLA-Cw4- or -Cw6-expressing cells was markedly decreased compared to the wild type-expressing cells. The ability to induce an inhibitory signal in the NK tumor line YTS transfected with KIR2DL1 was also impaired in the nonglycosylated mutant expressing cells. Furthermore, in a second functional assay, mutant HLA-Cw4 and -Cw6 molecules had impaired ability to induce signal transduction in BW cells expressing a KIR2DL1-CD3 zeta chain chimeric protein. Thus, the deletion of the N-linked glycosylation signal in HLA-Cw4 and -Cw6 greatly reduced recognition by KIR2DL1. Alternative interpretations of the data are discussed.

Galectin-3 promotes adhesion of human neutrophils to laminin.

Galectin-3 is a member of a growing family of animal lectins composed of three domains, with the amino-terminal half consisting of a short segment followed by tandem repeats, and the carboxyl-terminal half representing the carbohydrate-recognition domain. Previously, we have shown that galectin-3 binds to the surface of human neutrophils and is capable of activating these cells. We have now studied the effect of exogenous galectin-3 on adhesion of human neutrophils to laminin-coated microtiter plates and found that this lectin promotes the adhesion in a dose-dependent manner. The effect was dependent on the lectin's carbohydrate-binding function, as well as its amino-terminal region. The galectin-3-induced adhesion was reduced significantly in the presence of EDTA, even though Ca2+ and Mg2+ are not required for the lectin binding, and the adhesion was significantly less at 4 degrees C, as compared with 37 degrees C. Galectin-3 also induced neutrophil adhesion to fibronectin, which is not recognized by the lectin, but much higher concentrations of the lectin were required, and the effect is completely dependent on Ca2+ and Mg2+. We conclude that galectin-3 induces neutrophil adhesion to laminin through a combination of two distinct mechanisms: 1) the lectin bridges neutrophils to laminin, in a carbohydrate-dependent and Ca2+-, Mg2+-independent manner, and 2) the lectin induces activation of neutrophils, in the presence of the divalent cations, resulting in the positive regulation of other cell adhesion molecules and enhanced adhesion to laminin. The results suggest that galectin-3 may play a role in the traversing of neutrophils through the basement membrane at inflammation sites.