Unfolding of Plasmodium falciparum Triosephosphate Isomerase in Urea and Guanidinium Chloride: Evidence for a Novel Disulfide Exchange Reaction in a Covalently Cross-Linked Mutant†

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The C-m(urea)/C-m(GdmCl) ratio (where C-m is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide crosslinked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.

Reversible Swelling/Deswelling Characteristics of Ethylene Glycol Dimethacrylate Cross-Linked Poly(acrylic acid-co-sodium acrylate-co-acrylamide) Superabsorbents

Poly(acrylic acid-co-sodium acrylate-co-acrylamide) superabsorbent polymers (SAPs) cross-linked with ethylene glycol dimethacrylate (EGDMA) were synthesized by inverse suspension polymerization. The SAPs were swollen in DI water, and it was found that the equilibrium swelling capacities varied with the acrylamide content. The SAPs were subjected to reversible swelling/deswelling cycles in DI water and aqueous NaCl solution, respectively. The effect of the addition of an electrolyte on the swelling of the SAP was explored. The equilibrium swelling capacity of the SAPs was found to decrease with increasing concentration of added electrolyte in the swelling medium. The effect of the particle size of the dry SAPs on the swelling properties was also investigated. A first order model was used to describe the kinetics of swelling/deswelling, and the equilibrium swelling capacity, limiting swelling capacity, and swelling/deswelling rate coefficients were determined.

Formulation development and evaluation of lamivudine controlled release tablets using cross-linked sago starch

Objectives: Modified starches based polymeric substances find utmost applicability in pharmaceutical formulation development. Cross-linked starches showed very promising results in drug delivery application. The present investigation concerns with the development of controlled release tablets of lamivudine using cross-linked sago starch. Methods: The cross-linked derivative was synthesized with phosphorous oxychloride and native sago starch in basic pH medium. The cross-linked sago starch was tested for acute toxicity and drug-excipient compatibility study. The formulated tablets were evaluated for various physical characteristics, in vitro dissolution release study and in vivo pharmacokinetic study in rabbit model. Results: In vitro release study showed that the optimized formulation exhibited highest correlation (R) in case of zero order kinetic model and the release mechanism followed a combination of diffusion and erosion process. There was a significant difference in the pharmacokinetic parameters (T-max, C-max, AUC, V-d, T-1/2, and MDT) of the optimized formulation as compared to the marketed conventional tablet Lamivir (R). Conclusion: The cross-linked starch showed promising results in terms of controlling the release behavior of the active drug from the matrix. The hydrophilic matrix synthesized by cross-linking could be used with a variety of active pharmaceutical ingredients for making their controlled/sustained release formulations.

Cross-Linked, Biodegradable, Cytocompatible Salicylic Acid Based Polyesters for Localized, Sustained Delivery of Salicylic Acid: An In Vitro Study

In order to suppress chronic inflammation while supporting cell proliferation, there has been a continuous surge toward development of polymers with the intention of delivering anti-inflammatory molecules in a sustained manner. In the above backdrop, we report the synthesis of a novel, stable, cross-linked polyester with salicylic acid (SA) incorporated in the polymeric backbone and propose a simple synthesis route by melt condensation. The as-synthesized polymer was hydrophobic with a glass transition temperature of 1 degrees C, which increases to 17 degrees C upon curing. The combination of NMR and FT-IR spectral techniques established the ester linkages in the as-synthesized SA-based polyester. The pH-dependent degradation rate and the rate of release of salicylic acid from the as-synthesized SA-based polymer were studied at physiological conditions in vitro. The polyester underwent surface erosion and exhibited linear degradation kinetics in which a change in degradation rate is observed after 4-10 days and 24% mass loss was recorded after 4 months at 37 degrees C and pH 7.4. The delivery of salicylic acid also showed a similar change in slopes, with a sustained release rate of 3.5% in 4 months. The cytocompatibility studies of these polyesters were carried out with C2C12 murine myoblast cells using techniques like MTT assay and flow cytometry. Our results strongly suggest that SA-based polyester supports cell proliferation for 3 days in culture and do not cause cell death (<7%), as quantified by propidium iodide (PI) stained cells. Hence, these polyesters can be used as implant materials for localized, sustained delivery of salicylic acid and have applications in adjuvant cancer therapy, chronic wound healing, and as an alternative to commercially available polymers like poly(lactic acid) and poly(glycolic acid) or their copolymers.

Cross-linked Chitosan/Thermoplastic Starch Reinforced with Multiwalled Carbon Nanotubes Using Maleate Esters as Coupling Agent: Mechanical, Tribological and Thermal Characteristics

Bio-nanocomposites have been developed using cross-linked chitosan and cross-linked thermoplastic starch along with acid functionalized multiwalled carbon nanotubes (f-MWCNT). The nanocomposites developed were characterized for mechanical, wear, and thermal properties. The results revealed that the nanocomposites exhibited enhanced mechanical properties. The composites containing 3% f-MWCNT showed maximum compression strength. Tribological studies revealed that, with the addition of small amount of f-MWCNTs the slide wear loss reduced up to 25%. SEM analysis of the nanocomposites showed predominantly brittle fractured surface. Thermal analysis showed that the incorporation of f-MWCNTs has improved the thermal stability for the nanocomposites.

Long-Term Sustained Release of Salicylic Acid from Cross-Linked Biodegradable Polyester Induces a Reduced Foreign Body Response in Mice

There has been a continuous surge toward developing new biopolymers that exhibit better in vivo biocompatibility properties in terms of demonstrating a reduced foreign body response (FBR). One approach to mitigate the undesired FBR is to develop an implant capable of releasing anti-inflammatory molecules in a sustained manner over a long time period. Implants causing inflammation are also more susceptible to infection. In this article, the in vivo biocompatibility of a novel, biodegradable salicylic acid releasing polyester (SAP) has been investigated by subcutaneous implantation in a mouse model. The tissue response to SAP was compared with that of a widely used biodegradable polymer, poly(lactic acid-co-glycolic acid) (PLGA), as a control over three time points: 2, 4, and 16 weeks postimplantation. A long-term in vitro study illustrates a continuous, linear (zero order) release of salicylic acid with a cumulative mass percent release rate of 7.34 x 10(-4) h(-1) over similar to 1.5-17 months. On the basis of physicochemical analysis, surface erosion for SAP and bulk erosion for PLGA have been confirmed as their dominant degradation modes in vivo. On the basis of the histomorphometrical analysis of inflammatory cell densities and collagen distribution as well as quantification of proinflammatory cytokine levels (TNF-alpha and IL-1 beta), a reduced foreign body response toward SAP with respect to that generated by PLGA has been unambiguously established. The favorable in vivo tissue response to SAP, as manifest from the uniform and well-vascularized encapsulation around the implant, is consistent with the decrease in inflammatory cell density and increase in angiogenesis with time. The above observations, together with the demonstration of long-term and sustained release of salicylic acid, establish the potential use of SAP for applications in improved matrices for tissue engineering and chronic wound healing.

3D Macroporous Solids from Chemically Cross-linked Carbon Nanotubes

We demonstrate a new technique to generate multiple light-sheets for fluorescence microscopy. This is possible by illuminating the cylindrical lens using multiple copies of Gaussian beams. A diffraction grating placed just before the cylindrical lens splits the incident Gaussian beam into multiple beams traveling at different angles. Subsequently, this gives rise to diffraction-limited light-sheets after the Gaussian beams pass through the combined cylindrical lens-objective sub-system. Direct measurement of field at and around the focus of objective lens shows multi-sheet pattern with an average thickness of 7.5 μm and inter-sheet separation of 380 μm. Employing an independent orthogonal detection sub-system, we successfully imaged fluorescently-coated yeast cells (≈4 μm) encaged in agarose gel-matrix. Such a diffraction-limited sheet-pattern equipped with dedicated detection system may find immediate applications in the field of optical microscopy and fluorescence imaging. © 2015 Optical Society of America

Controlled Release of Salicylic Acid from Biodegradable Cross-Linked Polyesters

The purpose of this work was to develop a family of crosslinked poly(xylitol adipate salicylate)s with a wide range of tunable release properties for delivering pharmacologically active salicylic acid. The synthesis parameters and release conditions were varied to modulate polyester properties and to understand the mechanism of release. Varying release rates were obtained upon longer curing (35% in the noncured polymer to 10% in the cured polymer in 7 days). Differential salicylic acid loading led to the synthesis of polymers with variable cross-linking and the release could be tuned (100% release for the lowest loading to 30% in the highest loading). Controlled release was monitored by changing various factors, and the release profiles were dependent on the stoichiometric composition, pH, curing time, and presence of enzyme. The polymer released a combination of salicylic acid and disalicylic acid, and the released products were found to be nontoxic. Minimal hemolysis and platelet activation indicated good blood compatibility. These polymers qualify as ``bioactive'' and ``resorbable'' and can, therefore, find applications as immunomodulatory resorbable biomaterials with tunable release properties.

Architecture of Physalis Mottle Tymovirus as Probed by Monoclonal Antibodies and Cross-Linking Studies

Physalis mottle tymovirus (previously named belladonna mottle virus, Iowa strain) RNA was cross-linked to its coat protein by exposure of the intact virus to ultraviolet light. The site of cross-linking of the coat protein with the RNA was identified as Lys-10 by sequencing the oligonucleotide-linked tryptic peptide obtained upon HPLC separation subsequent to enzymetic digestion of the cross-linked and dissociated virus. Three monoclonal antibodies PA3B2, PB5G9, and PF12C9, obtained using denatured coat protein as antigen, cross-reacted effectively with the intact virus indicating that the epitopes recognized by these monoclonals are on the surface of the virus. Using the peptides generated by digestion with CNBr, clostripain, V-8 protease, or trypsin and a recombinant protein lacking the N-terminal 21 residues expressed from a cDNA clone, it was shown that PA3B2 recognizes the sequence 22-36 on the coat protein while PB5G9 and PF12C9 recognize region 75-110. These results suggest that Lys-10 is one of the specific sites through which the RNA interacts in the intact virus. The polypeptide segment (region 22-36) following this buried portion as well as the epitope within the region 75-110 are exposed in the intact virus. These observations are consistent with the canonical β-barrel structure observed in certain other plant viruses.

Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

Three-dimensional plotted hydroxyapatite scaffolds with predefined architecture: comparison of stabilization by alginate cross-linking versus sintering

Scaffolds for bone tissue engineering are essentially characterized by porous three-dimensional structures with interconnected pores to facilitate the exchange of nutrients and removal of waste products from cells, thereby promoting cell proliferation in such engineered scaffolds. Although hydroxyapatite is widely being considered for bone tissue engineering applications due to its occurrence in the natural extracellular matrix of this tissue, limited reports are available on additive manufacturing of hydroxyapatite-based materials. In this perspective, hydroxyapatite-based three-dimensional porous scaffolds with two different binders (maltodextrin and sodium alginate) were fabricated using the extrusion method of three-dimensional plotting and the results were compared in reference to the structural properties of scaffolds processed via chemical stabilization and sintering routes, respectively. With the optimal processing conditions regarding to pH and viscosity of binder-loaded hydroxyapatite pastes, scaffolds with parallelepiped porous architecture having up to 74% porosity were fabricated. Interestingly, sintering of the as-plotted hydroxyapatite-sodium alginate (cross-linked with CaCl2 solution) scaffolds led to the formation of chlorapatite (Ca9.54P5.98O23.8Cl1.60(OH)(2.74)). Both the sintered scaffolds displayed progressive deformation and delayed fracture under compressive loading, with hydroxyapatite-alginate scaffolds exhibiting a higher compressive strength (9.5 +/- 0.5MPa) than hydroxyapatite-maltodextrin scaffolds (7.0 +/- 0.6MPa). The difference in properties is explained in terms of the phase assemblage and microstructure.

Structural correlations in GexSe1-x glasses - a neutron diffraction study

Neutron diffraction measurement is carried out on GexSe1-x glasses, where 0.1 less than or equal to x less than or equal to 0.4, in a Q interval of 0.55-13.8 Angstrom(-1). The first sharp diffraction peak (FSDP) in the structure factor, S(Q), shows a systematic increase in the intensity and shifts to a lower Q with increasing Ge concentration. The coherence length of FSDP increases with x and becomes maximum for 0.33 less than or equal to x less than or equal to 0.4. The Monte-Carlo method, due to Soper, is used to generate S(Q) and also the pair correlation function, g(r). The generated S(Q) is in agreement with the experimental data for all x. Analysis of the first four peaks in the total correlation function, T(r), shows that the short range order in GeSe2 glass is due to Ge(Se-1/2)(4) tetrahedra, in agreement with earlier reports. Se-rich glasses contain Se-chains which are cross-linked with Ge(Se-1/2)(4) tetrahedra. Ge-2(Se-1/2)(6) molecular units are the basic structural units in Ge-rich, x = 0.4, glass. For x = 0.2, 0.33 and 0.4 there is evidence for some of the tetrahedra being in an edge-shared configuration. The number of edge-shared tetrahedra in these glasses increase with increasing Ge content.

Mechanism of coupling periodate-oxidized nucleosides to proteins

This paper describes a mechanism of coupling periodate-oxidized nucleosides to proteins. Each of the dialdehyde groups of a periodate-oxidized nucleoside is shown to couple to lysine residues on different protein molecules through Schiff bases, thereby cross-linking different protein molecules, forming a polymer. This is in contrast to the previous model in which nucleosides were suggested to couple to proteins through a morpholine structure. The cross-linked structure of the nucleoside-antigen, significantly different when compared to the native protein, may affect the specificity and the efficiency of antibody production.