Biophysical analysis of the molecular interactions between polysaccharides and mucin.

Mucoadhesive materials adhere persistently to mucosal surfaces. A mucoadhesive delivery system could therefore facilitate the controlled release of drugs and optimize their bioavailability in mucosal tissues. Polysaccharides are the most versatile class of natural polymers for transmucosal drug delivery. We used microviscosimetry to explore the mucoadhesion of a library of polysaccharide families with diverse structural characteristics as a first step toward the rational design of mucoadhesive polysaccharide-based nanoformulations. Here we show that the magnitude of deviation between the viscosity of mixed polysaccharide-mucin solutions and the corresponding individual stock solutions can indicate underlying molecular interactions. We found that nonlinear monotonic curves predicted a correlation between the magnitude of interaction and the ability of polysaccharide coils to contract in the presence of salt (i.e., chain flexibility). Charge-neutral polysaccharides such as dextran and Streptococcus thermophilus exopolysaccharide did not interact with mucin. Synchrotron small-angle X-ray scattering (SAXS) data supported the previously described structural features of mucin. Furthermore, high-q scattering data (i.e., sensitive to smaller scales) revealed that when mucin is in dilute solution (presumably in an extended conformation) in the presence of low-Mw alginate, its structure resembles that observed at higher concentrations in the absence of alginate. This effect was less pronounced in the case of high-Mw alginate, but the latter influenced the bulk properties of mucin-alginate mixtures (e.g., hydrodynamic radius and relative viscosity) more prominently than its low-Mw counterpart.

Effect of peptide and guest charge on the structural, mechanical and release properties of ß-sheet forming peptides.

The effect of peptide charge on the self-assembly, gelation behavior, and model drug release profiles has been explored here for three octa-peptides, VEVKVEVK (VEK2), VKVKVEVK (VEK3), and VEVEVKVE (VEK1), that carry a net charge of 0, +2, and -2 at neutral pH, respectively. Transparent, self-supporting hydrogels were found to form above a critical concentration when the peptide charge modulus was >1 and this was independent of the sign of the charge. TEM, SAXS, and shear rheology revealed that there were no differences in hydrogel structure or mechanical properties when the peptides were at the same concentration and carried the same charge modulus. All peptides were found to form dense fibrillar networks formed by ß-sheet rich single fibers where lateral aggregation of the fibers occurred and increased with decreasing charge modulus. Such behavior was found to correlate with an increase in hydrogel mechanical properties, demonstrating that fiber lateral aggregation is inextricably linked with the mechanical properties of these hydrogels. Two hydrophilic model drug molecules, namely napthol yellow (NY) and martius yellow (MY), were subsequently incorporated within the VEK1 and VEK3 hydrogels at pH 7 and although they did not effect the self-assembly of the peptide at a molecular level, they did effect the level of lateral fiber aggregation observed and, therefore, the mechanical properties of the hydrogels. The release of each molecule from the hydrogels was monitored over time and shown to be controlled by Fickian diffusion where the diffusion rate, D, was dependent on the ratio between the overall effective charges carried by the peptide, i.e., the fibrillar network, and the overall charges carried by the guest molecules, but independent from the hydrogel concentration and mechanical properties within the ranges investigated. This work highlights the possibility of controlling the rate of release of small drug molecules by manipulating the charges on the guest molecules as well as the charged state of the self-assembling peptide.

Hybrid thermoreversible gels from covalent polymers and organogels.

This paper reports on experiments intended for investigating the feasibility of preparing hybrid thermoreversible gels from covalent polymers and noncovalent self-assembling pi-conjugated molecules. The formation and the degree of dispersion of these hybrid gels have been studied with polystyrenes of various tacticities and oligo(p-phenylenevinylene) molecules (OPV) in different nonpolar organic solvents. Detailed investigations of the systems have been carried out by DSC, SAXS, and AFM. It is shown that no liquid-liquid phase separation is involved, indicating that the systems are highly compatible, and that the growth of one type of gel does not interfere with the other. These studies reveal that the resultant hybrid gels are composed of the intermingled fibrillar architectures of both gels.

Aging and yielding in a sheared AOT/iso-octane/water lyotropic lamellar phase.

We define a creep-flow-based measurement procedure to allow reliable and reproducible results on aging and yielding materials to be obtained. Investigation of the effects of different parameter such as the pre-shear time, the recovery time and the applied stress magnitude on the viscoelastic properties of a lyotropic liquid crystal phase is reported. Cryo-TEM observations indicate the formation of multiconnected bilayers at rest. Shearing the investigated material shows a propensity to acquire all the macroscopic properties of "soft jammed systems". These properties are then interpreted in terms of shear-induced structural rearrangement on the basis of cryofracture observation obtained at different times after the preshear imposed.

BAFF and rheumatic autoimmune disorders: implications for disease management and therapy.

Interest in B-cells has been revived due to the description of new functions. Supporting a role for B-cells in the genesis of autoimmune diseases is the fact that the B-cell activating factor of the TNF ligand family (BAFF) is essential in their physiology. However, in each disease, this is restricted to a subgroup of patients. Based on experiments in mice, and validated in humans, this new cytokine has been highlighted. Excessive production of BAFF alters immune tolerance by rescuing self-binding B-cells. Overexpression in mice leads to autoimmune manifestation, and BAFF levels are elevated in the serum of autoimmune patients. Similar abnormalities occur in chronic lymphocytic leukemia. Recent works suggest that antagonizing the protein (or competing for its receptors) is relevant to the treatment. Advances in our understanding of the BAFF system offers the opportunity to improve our therapeutic approach.

Self-assembly of maltoheptaose-b-PMMA block copolymer systems: 10nm Resolution in thin film and bulk states.

This paper describes the self-assembly of oligosaccharide-based hybrid block copolymers (BCPs) consisting of maltoheptaose (MH) and poly(methyl methacrylate) (PMMA) into 10nm scale lamellar and cylindrical phases depending on the volume fractions of MH (ϕMH) and the annealing process. Time resolved SAXS study of the BCP bulk samples during thermal annealing indicated that the BCPs phase separate into 10nm scale periodical structures. The solvent vapor annealing induced self-organizations of the BCP into different phases depending on ϕMH and the weight fraction of THF/H2O. BCPs with relatively higher ØMH, MH-b-PMMA3k (ϕMH=0.27) and MH-b-PMMA5k (ϕMH=0.16) self-organized into lamellar phases while the BCP sample with relatively lower ϕMH, MH-b-PMMA9k (ϕMH=0.10), self-organized into cylindrical phase by using THF/H2O=1/4 (w/w). On the other hand, the solvent vapor annealing with larger fraction of THF, i.e. THF/H2O=2/3 (w/w), induced cylindrical phases for MH-b-PMMA3k and MH-b-PMMA5k.

87Rb+ spin relaxation in enzymically purified and in untreated iota-carrageenan.

The temperature dependences of the transverse (R2) and longitudinal (R1) n.m.r. relaxation rates of 87Rb+ in aqueous 5% iota-carrageenan have been compared with similar data for a sample purified by treatment with kappa-carrageenase. In each sample, the relaxation rates were sensitive to the conformation (helix or random coil). In the intact sample, the small (less than or equal to 5%) fraction of kappa-carrageenan (which, in its helical state, specifically binds rubidium ions) was solely responsible for the pronounced line-broadening that has been observed hitherto for 87Rb in iota-carrageenan gels. In the purified sample, the effects on the relaxation of 87Rb induced by iota-carrageenan are similar to those found in comparable systems in the absence of site-binding of the ions. Thus, there was a modest enhancement of the relaxation with R1 approximately R2 for the flexible coil conformation and a comparably larger effect, with significant contributions from dynamic processes on the time-scale of the inverse resonance frequency or longer, for the thicker, more highly charged and rigid helix conformation of iota-carrageenan.

NMR spectroscopic investigation of agarose oligomers produced by an alpha-agarase.

The 13C NMR signals of various even and odd agarose oligosaccharides with either D-galactose or 3,6-anhydro-alpha-L-galactose at the reducing end have been assigned. The chemical shifts in water of the agaro- and the neoagaro-oligosaccharides are compared and the influence of dimethyl sulfoxide on the chemical structure of the agaro-oligosaccharides is reported. The 3,6-anhydro-L-galactose residue at the reducing end of agaro-oligosaccharides is in the hydrated form.

NMR spectroscopy analysis of oligoguluronates and oligomannuronates prepared by acid or enzymatic hydrolysis of homopolymeric blocks of alginic acid. Application to the determination of the substrate specificity of Haliotis tuberculata alginate lyase.

The 1H and 13C NMR chemicals shifts of the various saturated and unsaturated timers obtained by acid or enzymatic depolymerisation of homopolymeric blocks of alginates are reported. In addition, 13C NMR chemical shifts are assigned for several saturated oligomers of higher polymerisation degrees. Breakdown of alginate and of homopolymeric alginate blocks by Haliotis tuberculata alginate lyase was monitored with 1H NMR spectroscopy and the signals relevant to the identification of the lyase products are pointed out. The enzymes performs beta-elimination on the mannuronic acid residues, independently of their immediately surrounding neighbours. Application of this approach to the analysis of the substrate specificity of alginate lyases is discussed.

On the occurrence of ternary complexes in agarose gels as studied by the contrast variation method.

The contrast variation method by isotopic labelling as used in small-angle neutron scattering experiments is applied to the case of agarose systems (sol and gels) in order to cast some light on the occurrence of complexes (crystallosolvates). This approach proves to be helpful for giving further support to the existence of ternary complexes in agarose/water/DMSO gels and in agarose/DMSO sols. The results are discussed in the light of temperature-composition phase diagrams established previously.

Agarose chain conformation in the sol state by neutron scattering.

We report on small-angle neutron scattering investigations into the agarose conformation in water. We show that at 70 degrees C in the sol state agarose chains are fairly rigid with a lower limiting value for the persistence length of about 9 nm and a mass per unit length of muL = 360 +/- 36 g/mol x nm. The value of the latter parameter is consistent with single-stranded helices as those proposed recently by Foord and Atkins. Such a high rigidity together with such a low linear mass leads one to wonder how would the chains manage to intertwine should they form double helices in the gel state.

N.m.r. studies of synergistic kappa carrageenan-carob galactomannan gels.

C-n.m.r. spectroscopy has been used to investigate the carob galactomannan-kappa carrageenan binary gels. Starting from partially depolymerized carob samples, evidence for interaction and intermolecular binding was found by analysis of the spectra obtained in quantitative conditions and in the absolute intensity mode. From these, a reconstitution of the signals corresponding to C-1, C-4, C-5 and C-6 showed the existence of three kinds of galactomannan chains; the first ones with a low galactose content were strongly connected and as such had completely lost their mobility; but the chains with a high galactose content were still detected and could be divided in two groups according to their mobility.

Modelling studies of solvent effects on the conformational stability of agarobiose and neoagarobiose and their relationship to agarose.

The conformational equilibria of the low energy conformers of the repeating units of agarose, i.e. neoagarobiose and agarobiose have been studied theoretically in five solvents. The structure of each individual conformer has been refined by molecular mechanics calculations, from the distinct low-energy regions determined from potential energy surfaces. The stability of the conformers in dilute solution has been evaluated through consideration of the relaxed intramolecular energy including electrostatic, dispersion and cavity terms. For each disaccharide, the calculated abundance of conformers is practically independent of the solvent. Dipole moments and linkage rotation have been calculated for each of the low energy conformers. The calculated molar fractions of the stable conformers were used to model the optical rotation values measured for a single strand of agarose chain in its disordered state, and a satisfactory agreement has been reached (lambda cal = -133 degrees, lambda obs = -145 degrees). For the agarose molecule in the solid state, a left-handed threefold single helix, repeating in 2.85 nm is predicted. Such a helix, which represents the calculated stable conformation for a single strand of agarose, should be considered in descriptions of the molecular basis of sol-gel transition of agarose.

Synergy of the kappa-carrageenan-carob galactomannan blend inferred from rheological studies.

Rheological measurements have been used to investigate kappa-carrageenan-carob galactomannan mixed gels at different ratios, in particular for very low kappa-carrageenan concentrations. The viscoelastic properties of the gels were explored using sinusoidal tests on dynamic rheometers and compression tests between parallel plates. The properties of the binary gels were compared with the kappa-carrageenan gels and with the carob galactomannan solutions. These measurements display significant synergy for very low kappa-carrageenan concentrations. From these data, it appears that the synergy is based on a gel structure defined by a coupled network with specific junction zones. However, above a total polysaccharide concentration of 8-10 g l-1, self-association of galactomannan chains could also take place.

Polyelectrolyte microstructure in chitosan aqueous and alcohol solutions.

This work deals with chain ordering in aqueous and water-alcohol solutions of chitosan. The so-called polyelectrolyte peak is investigated by small-angle synchrotron X-ray scattering. The polyelectrolyte microstructure was characterized by the position of the maximum of the polyelectrolyte scattering peak qmax, which scales with the polymer concentration cp as qmax approximately cp alpha. An evolution of the power law exponent alpha is observed as a function of the degree of acetylation (DA) of chitosan, which is responsible for changes of both the charge density (f) and the hydrophobicity of the polymer chains. The results highlighted the two organization regimes of the theory of Dobrynin and Rubinstein, investigated here for the first time for a natural polymer. At low DAs, alpha approximately 1/2, in agreement with a pearl necklace organization where the structure is controlled by the string between pearls. For higher DA, alpha approximately 1/3, and the correlation revealed by the polyelectrolyte peak is controlled by the pearls. This analysis offers a way to study quantitatively the balance between solvophobic-solvophilic interactions that play an important role in the solution properties of natural polymers. In addition, the role of several parameters acting on the interaction balance were evidenced, such as the nature of the counterion, the composition of the solvent (amount of alcohol in the aqueous solution), and the screening of Coulombic forces by salt addition. Finally, the nanostructure transition from a polyelectrolyte solution to a physical gel is discussed. The gel state is reached when the solvophobic interactions are favored, but depending on the gelation route the polyelectrolyte ordering could be preserved or not.

Central and peripheral RAG protein re-expression: underestimate mechanisms of tolerance?

The generation of developing B cells in the bone marrow is regulated by recombination activating genes RAG1 and RAG2 proteins. They contribute to the synthesis of functional antibodies (Abs) that can present self-reactivities following V(D)J (V, variable; D, diversity and J, joining) recombination. The emergence of autoreactive B cells is prevented by deletion through apoptosis, by stimulation blockade through anergy, or by synthesis of a new B-cell receptor through receptor edition. In the periphery, somatic hypermutation during the course of germinal centre (GC) responses can lead to the appearance of autoreactive and low-affinity Ab-producing B cells. Apoptotic deletion and receptor revision regulate these autoreactive and inappropriate B cells. Moreover, the presence of RAG-positive B cells outside GCs suggest that still uncharacterized regulation checkpoint, associated with secondary V(D)J recombination, also contribute to the regulation of autoreactivities. Failure in central and/or peripheral tolerance mechanisms associated with RAG expression could contribute to the terminal differentiation of autoreactive B cells leading to autoimmune states.

Computer modeling of polysaccharide-polysaccharide interactions: an approach to the kappa-carrageenan-mannan case.

A computer program SAINT has been developed for the investigation of the structure and for the prediction of minimum-energy structure of polysaccharide-polysaccharide complexes. The energy minimization is carried out on internal geometrical parameters--namely bond angles, torsional angles, and five parameters describing the mutual orientations of polysaccharide chains. For this purpose, the nonderivative method of conjugated directions is used. This procedure was applied to computer modeling of an idealized model of the binary gelling kappa-carrageenan and galactomannan system. It is shown that the interaction between two chains influences the structure of the individual polysaccharide molecule and that in the minimum-energy structures of the complex, the conformation of the chains does not correspond to the lowest energy.

Computer modelling of kappa carrageenan-mannan interactions.

Molecular modelling has been used as a theoretical approach to investigate the kappa carrageenan structure and its interactions with mannan chains. Calculations revealed the existence of six minima for the kappa carrageenan structure in solution. Two of them were very close to the structure found in the solid state. The methodology allowed the calculation of a theoretical counterpart of the structures based on x-ray fibre diffraction studies. In the second step of this study, we have shown that there is the possibility of interactions between kappa carrageenan double helices and mannan chains. This interacting process is allowed by the flexibility of the mannan chains and structural changes of the kappa carrageenan double helices. The calculations suggest that a disaccharide mannan fragment might be required for recognition. The results of our investigation are in good agreement with a model of gel structure based on experimental data. This approach could be applied to simulate and predict other associations in molecular assemblies.

Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B.

The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S. Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207-212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An alpha-agarase, i.e. specific for the alpha(1-->3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatography (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified alpha-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 degrees C or by removing calcium. In addition, a beta-galactosidase specific for the end products of the alpha-agarase was present in the alpha-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.

Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium.

A bacterial strain able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. From the cell-free supernatant of cultures grown on crude lambda-carrageenan, a kappa-carrageenase was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S 200 HR and ion-exchange chromatography on DEAE--Sepharose-CL6B. The purified kappa-carrageenase was detected as a single protein upon SDS/PAGE. Its molecular mass was estimated at 40 kDa. Activity was observed against kappa-carrageenan over the pH range 5.0-8.5 and was optimal at pH 7.2 in Tris buffer or 7.0 in Mops buffer. The enzyme activity remained stable at 30 degrees C, but only for up to 1 h at 40 degrees C. Analysis of the degradation products of the kappa-carrageenase by gel filtration and 13C-NMR spectroscopy indicated that the enzyme degrades kappa-carrageenan down to the level of the kappa-neocarratetraose sulfate. The properties of this new enzyme are compared with those of previously characterized carrageenases.