T1-Weight Magnetic Resonance Imaging Performances of Iron Oxide Nanoparticles Modified with a Natural Protein Macromolecule and an Artificial Macromolecule

To optimize the iron oxide nanoparticles as T1-weight contrast for in vivo magnetic resonance imaging (MRI), numbers of macromolecule ligands have been explored with considerable effort. However, reports refer to the comparison of the T1-weight contrast performances of iron oxide nanoparticles modified with natural and artificial macromolecule ligands are still limited. In this work, we used a typical natural protein macromolecule (bovine serum albumin, BSA) and an artificial macromolecule (poly(acrylic acid)-poly(methacrylic acid), PMAA-PTTM) as surface ligands to fabricate Fe3O4-BSA and Fe3O4-PMAA-PTTM nanoparticles with similar size and magnetization by the coprecipitation method and compared their MRI performances. In vitro and in vivo experiments revealed that Fe3O4-BSA with lower cytotoxicity exhibited higher r2/r1 ratio in solution and darkening contrast enhancement for liver and kidney sites of mice under T1-weight imaging, while Fe3O4-PMAA-PTTM displayed much lower r2/r1 ratio in solution and brighter contrast enhancement for liver and kidney sites. These remarkably different MRI behaviors demonstrated that the surface ligands play an important role for optimizing the MRI performance of Fe3O4 nanoparticles. We expect these results may facilitate the design of macromolecule ligands for developing an iron oxide–based T1-weight contrast agent.

Interaction of the Fungicide Tebuconazole with Human Serum Albumin: A Preliminary Study

The interactions between the fungicide tebuconazole and human serum albumin were investigated using fluorescence and circular dichroism spectroscopies. The experimental results showed that the fluorescence quenching of the protein by the tebuconazole molecule was a result of the formation of a ligand-protein complex with a binding constant of 8.51×103 l.mol−1 and the number of binding sites in the macromolecule was close to 1. These findings demonstrated the fact that although the binding affinity of tebuconazole to the protein may be slight, it was very similar to other triazole fungicides. In addition, tebuconazole stabilized the α-helical secondary structure of the human serum albumin due to the increase of the α-content in the protein macromolecule.

Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity

Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginaseto resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

Comparison of Antigenic Regions Identified on IgG1Fc Using Bioinformatics vs Pepscan Analysis

Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identified. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents. As an extension to this work we have used these peptides to evaluate the location of epitope targets of five IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294-EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and five epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epitopes in the CH2 domain (256-TPEVTCVVVDVSHED-270) and CH3 domain (418-QQGNVFSCSVMHEAL-432). Similarly, RFAb PRTS1 detected all four epitopes plus a fifth in the CH3 domain (382-ESNGQPENNYKTTPP-396). In essence there was a consensus of target epitopes identified by these rheumatoid factor antibodies. Interestingly, two epitopes (256–270, CH2 domain and 360–374, CH3 domain) were novel in that they had not been identified in previous pepscan studies. The other epitopes recognised, either overlapped or were immediately adjacent to previous epitopes detected by poly/monoclonal rheumatoid factor antibodies. Molecular modelling (PCImdad) of IgG1Fc showed that all five epitopes were exposed and surface accessible for antibody interaction. In addition, a bioinformatics analysis of the Fc region using ExPASy was employed to identify key antigenic determinants. This ‘in silico’ approach may provide a means of determining key regions without the need to develop overlapping peptides spanning the entire length of a macromolecule.