Synthesis of folate- pegylated polyester nanoparticles encapsulating ixabepilone for targeting folate receptor overexpressing breast cancer cells.

The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)-functionalized poly(ethylene glycol)-poly(propylene succinate) (PEG-PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by (1)H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC-UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5%) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA-PEG-PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG-PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA-PEG-PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor.

Biophysical characterization of the influence of salt on tetrameric SecB.

SecB is a tetrameric chaperone, with a monomeric molecular mass of 17 kDa, that is involved in protein translocation in Escherichia coli. It has been hypothesized that SecB undergoes a conformational change as a function of the salt concentration. To gain more insight into the salt-dependent behavior of SecB, we studied the protein in solution by dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and small angle neutron scattering. The results clearly demonstrate the large influence of the salt concentration on the behavior of SecB. At high salt concentration, SecB is a non-spherical protein with a radius of gyration of 3.4 nm. At low salt concentration the hydrodynamic radius of the protein is apparently decreased, whereas the ratio of the frictional coefficients is increased. The protein solution behaves in a non-ideal way at low salt concentrations, as was shown by the analytical ultracentrifugation data and a pronounced interparticle effect observed by small angle neutron scattering. A possible explanation is a change in surface charge distribution dependent on the salt concentration in the solvent. We summarize our data in a model for the salt-dependent conformation of tetrameric SecB.

Crystallization and preliminary X-ray analysis of Drosophila glutathione S-transferase-2.

The sigma-class glutathione S-transferase-2 (GST-2) from Drosophila melanogaster is predominantly found within the indirect flight muscles (IFMs), where it is bound to the 'heavy' subunit of the IFM thin filament troponin complex (Tn-H). An N-terminal extension found in GST-2 is unique within the sigma GST class and may be involved in its interaction with Tn-H or modulate its enzymatic function. The recombinant protein has been crystallized at room temperature using ammonium sulfate as precipitant. Synchrotron radiation was used to measure a complete native data set to 1.75 A resolution from flash-cooled crystals. The crystals belong to one of the trigonal space groups P3(1)21 or P3(2)21, with unit-cell parameters a = b = 89.7, c = 131.8 A. The self-rotation function is consistent with a GST-2 dimer in the asymmetric unit.

Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14.

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.

The glutamine-rich domain of the Drosophila GAGA factor is necessary for amyloid fibre formation in vitro, but not for chromatin remodelling.

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.

A Triclinic Crystal Form of the Lectin Concanavalin A

The molecular structure of a triclinic crystal form of concanavalin A has been refined at 2.4 A resolution. The crystals have unit cell dimensions a = 78.8 A, b = 79.3 A, c = 133.3 A, alpha = 97.1degrees, beta = 90.2degrees, and gamma = 97.5degrees and contain two tetramers per asymmetric unit each with approximate 222 symmetry. The final crystallographic R-factor is 0.205 and the free-R-factor is 0.265 in the resolution range 6.0 to 2.4 A. The conformation of the tetramer is more similar to that found in concanavalin A saccharide complexes than in the previously reported I222 crystal form of uncomplexed concanavalin A. A comparison of the molecular packing between the two crystal forms shows a more open arrangement with large solvent channels through the crystal.

The crystal structure of the complexes of concanavalin A with 4'-nitrophenyl-alpha-D-mannopyranoside and 4'-nitrophenyl-alpha-D-glucopyranoside.

Concanavalin A (Con A) is the best-known plant lectin and has important in vitro biological activities arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complexes of Con A with 4'-nitrophenyl-alpha-D-mannopyranoside (alpha-PNM) and 4'-nitrophenyl-alpha-D-glucopyranoside (alpha-PNG) have been crystallized in space group P2(1)2(1)2 with cell dimensions a = 135.19 A, b = 155.38 A, c = 71.25 A and a = 134.66 A, b = 155.67 A, and c = 71.42 A, respectively. X-ray diffraction intensities to 2.75 A for the alpha-PNM and to 3.0 A resolution for the alpha-PNG complex have been collected. The structures of the complexes were solved by molecular replacement and refined by simulated annealing methods to crystallographic R-factor values of 0.185/0.186 and free-R-factor values of 0.260/0.274, respectively. In both structures, the asymmetric unit contains four molecules arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site near the surface of each monomer. The nonsugar (aglycon) portion of the compounds used helps to identify the exact orientation of the saccharide in the sugar-binding pocket and is involved in major interactions between tetramers. The hydrogen bonding network in the region of the binding site has been analyzed, and only minor differences with the previously reported Con A-methyl-alpha-D-mannopyranoside complex structure have been observed. Structural differences that may contribute to the slight preference of the lectin for mannosides over glucosides are discussed. Calculations indicate a negative electrostatic surface potential for the saccharide binding site of Con A, which may be important for its biological activity. It is also shown in detail how a particular class of hydrophobic ligands interact with one of the three so-called characteristic hydrophobic sites of the lectins.