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.

Cyclometalated complexes derived from calix[4] arene bisphosphites and their catalytic applications in cross-coupling reactions

Palladium and platinum dichloride complexes of a series of symmetrically and unsymmetrically substituted 25,26;27,28-dibridged p-tert-butyl-calix[4]arene bisphosphites in which two proximal phenolic oxygen atoms of p-tert-butyl-or p-H-calix[4]arene are connected to a P(OR) ( R = substituted phenyl) moiety have been synthesized. The palladium dichloride complexes of calix[4]arene bisphosphites bearing sterically bulky aryl substituents undergo cyclometalation by C-C or C-H bond scission. An example of cycloplatinated complex is also reported. The complexes have been characterized by NMR spectroscopic and single crystal X-ray diffraction studies. During crystallization of the palladium dichloride complex of a symmetrically substituted calix[4]arene bisphosphite in dichloromethane, insertion of oxygen occurs into the Pd-P bond to give a P,O-coordinated palladium dichloride complex. The calix[4]arene framework in these bisphosphites and their metal complexes adopt distorted cone conformation; the cone conformation is more flattened in the metal complexes than in the free calix[4]arene bisphosphites. Some of these cyclometalated complexes proved to be active catalysts for Heck and Suzuki C-C cross-coupling reactions but, on an average, the yields are only modest. (C) 2011 Elsevier B.V. All rights reserved.

Pd-Catalyzed Cross-Coupling Reactions of Hydrazones: Regioselective Synthesis of Highly Branched Dienes

The regioselective formation of highly branched dienes is a challenging task. Design and exploration of alternative working models to achieve such a regioselectivity to accomplish highly branched dienes is considered to be a historical advancement of Heck reaction to construct branched dienes. On the basis of the utility of carbene transfer reactions, in the reaction of hydrazones with Pd(II) under oxidative conditions, we envisioned obtaining a Pd-bis-carbene complex with alpha-hydrogens, which can lead to branched dienes. Herein, we report a novel Pd-catalyzed selective coupling reaction of hydrazones in the presence of t-BuOLi and benzoquinone to form the corresponding branched dienes. The utility of the Pd catalyst for the cross-coupling reactions for synthesizing branched conjugated dienes is rare. The reaction is very versatile and compatible with a variety of functional groups and is useful in synthesizing heterocyclic molecules. We anticipate that this Pd-catalyzed cross-coupling reaction will open new avenues for synthesizing useful compounds.

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.

Role of transglutaminase-mediated cross-linking of protein in the temperature-dependent setting of Alaska pollack surimi

During the low temperature setting of fish paste, myosin heavy chain (MHC) is polymerized to cross-linked myosin heavy chain (CMHC), which is considered to occur by the action of endogenous transglutaminase (TGase). In this study the contribution of TGase on the setting of Alaska pollack surimi at different temperatures was studied. Alaska pollack surimi was ground with 3% NaCl, 30% h2o and with or without ethylene glycol bis (β-aminoethylether) N, N, N¹,N¹- tetra acetic acid (EGTA), an inhibitor of TGase. Among the pastes without EGTA, highest TGase activity was observed at 25°C but breaking force of the gel set at 25°C was lower than that set at 30°, 35°, and 40°C. Addition of EGTA (5m mol/kg) to the paste suppressed TGase activity at all setting temperatures from 20° to 40°C. Gelation of the pastes and cross-linking of MHC on addition of EGTA were suppressed completely at 20° and 25°C, partially at 30° and 35°C, and not at all at 40°C. The findings suggested that during the setting of Alaska pollack surimi TGase mediated cross-linking of MHC was strong at around 25°C but the thermal aggregation of MHC by non-covalent bonds was strong at above 35°C. Setting of surimi at 40°C and cross-linking of its MHC did not involve TGase.

Electroactive Aniline Pentamer Cross-Linking Chitosan for Stimulation Growth of Electrically Sensitive Cells

A new kind of electroactive polymers was synthesized by using aniline pentamer (AP) cross-linking chitosan (CS) in acetic acid/DMSO/DMF solution. UV-vis and CV confirmed the electroactivity of polymers in acidic aqueous solution. The amphiphilic polymers self-assembled into 200-300 nm micelles by dialysis against deionized water from the acetic acid buffer solution. Three samples with different weight percentages of AP were used to identify the relationship between the content of AP and the differentiation of rat neuronal pheochromocytoma PC-12 cells without external stimulation.

Immobilization of dipyridyl complex to magnetic nanoparticle via click chemistry as a recyclable catalyst for Suzuki cross-coupling reactions

A magnetic nanoparticle (MNP)-supported di(2-pyridyl)methanol palladium dichloride complex was prepared via click chemistry. The MNP-supported catalyst was evaluated in Suzuki coupling reaction in term of activity and recyclability in DMF. It was found to be highly efficient for Suzuki coupling reaction using aryl bromides as substrates and could be easily separated by an external magnet and reused in five consecutive runs without obvious loss of activity.

Synthesis of fluorene-based oligomeric organoboron reagents via Kumada, Heck, and stille cross-coupling reactions

Boronic pinacol ester group is not reactive in Kumada, Heck and Stille coupling reaction conditions. Fluorene-based sophisticated organoboron compounds were synthesized by means of Palladium catalyzed Kumada, Heck and Stille cross-coupling reactions from halofluorenyl boronic esters.

Effect of cross-linking of high-density polyethylene. I. On spherulitic structures

Crystallization behavior and spherulitic structure of linear high-density polyethylene (HDPE), after being irradiated in its molten state by gamma -rays, was investigated by small-angle laser scattering (SALS) and differential scanning calorimetry (DSC). Significant changes in the crystallization of HDPE during cooling in air before and after being irradiated in the melt were observed. A critical minimum average molar mass between cross-links (200 carbon-carbon bonds) for spherulite formation in such an irradiated HDPE network was obtained.

Advanced research work on radiation cross-linking of polymers in CIAC

Recent research carried out at the Chinese Institute of Applied Chemistry has contributed significantly to the understanding of the radiation chemistry of polymers. High energy radiation has been successfully used to cross-link fluoropolymers and polyimides. Here chain flexibility has been shown to play an important role, and T-type structures were found to exist in the cross-linked fluoropolymers. A modified Charlesby-Pinner equation, based upon the importance of chain flexibility, was developed to account for the sol-radiation dose relationship in systems of this type. An XPS method has been developed to measure the cross-linking yields in aromatic polymers and fluoropolymers, based upon the dose dependence of the aromatic shake-up peaks and the F/C ratios, respectively. Methods for radiation cross-linking degrading polymers in polymer blends have also been developed, as have methods for improving the radiation resistance of polymers through radiation cross-linking.

RADIATION CROSS-LINKING OF POLYTETRAFLUOROETHYLENE

Polytetrafluoroethylene (PTFE) is considered to be a typical radiation degradative polymer. In this work the polymer was crosslinked by means of radiation at 335 +/- 5-degrees-C under vacuum. Crosslinked PTFE showed a great improvement in high temperature

EFFECTS OF STRUCTURE MULTIPLICITY ON MECHANISM OF RADIATION CROSS-LINKING OF POLYMERS

In an attempt to explore the effects of structural multiplicity of polymers on the mechanism of radiation crosslinking, the adaptability of the Charlesby-Pinner's equation and its various modified versions are examined. It is recognized that both chemical