Screening, characterization and specific binding mechanism of aptamers against human plasminogen Kringle 5.

Plasminogen Kringle 5 is one of the most potent cytokines identified to inhibit the proliferation and migration of vascular endothelial cells. Herein, six aptamer candidates that specifically bind to Kringle 5 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of screening against Kringle 5, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. The preferred aptamer KG-4, which demonstrated a low dissociation constant (Kd) of âˆ¼ 432 nM and excellent selectivity for Kringle 5. A conserved "motif" of eight bases located at the stem-loop intersection, common to the aptamer, was further confirmed as the recognition element for binding with Kringle 5. The bulge formed by the motif and depression on the lysine binding site of Kringle 5 were both located at the binding interface, and the "induced fit" between their structures played a central role in the recognition process. Kringle 5 interacts KG-4 primarily through enthalpy-driven van der Waals forces and hydrogen bond. The key nucleotides A34 and C35 at motif on KG-4 and the positively charged amino acids in the loop 1 and loop 4 regions on Kringle 5 play a major role in the interaction. Furthermore, KG-4 dose-dependently reduced the proliferation inhibition of vascular endothelial cells by Kringle 5 and had a blocking effect on the function of Kringle 5 in inhibiting migration and promoting apoptosis of vascular endothelial cells in vitro. This study put a new light on protein-aptamer binding mechanism and may provide insight into the treatment of ischemic diseases by target depletion of Kringle 5.

Molecular recognition and binding of CcrA from Bacteroides fragilis with cefotaxime and ceftazidime by fluorescence spectra and molecular docking.

In search of new super-bacterial inhibitor agents, the recognition and binding mechanism of the B1 subclass MßL CcrA from Bacteroides fragilis with cefotaxime (CTX) and ceftazidime (CAZ) were studied using spectroscopy analysis and molecular docking. The results showed that the fluorescence quenching of CcrA induced by CTX and CAZ were all due to the complex formation, which belonged to static quenching and was forced by hydrogen bonds and Van der Waals forces, despite the greater binding ability of CTX with CcrA than CAZ. Upon recognizing CTX or CAZ, the CcrA opened its binding pocket by the microenvironmental and conformational of three loops changing to promote an induced-fit of the freshly introduced antibiotics. In addition, the whole antibiotic molecule ultimately entered the active pocket of CcrA with its original carbonate replaced by the carboxyl oxygen of the hexatomic ring adjacent to the ß-lactam ring in CTX or CAZ, forming a new tetrahedral coordination structure at the Zn2 site. Moreover, the difference in steric hindrance and electrostatic effects of the side chain affected the binding ability of the two antibiotics to the CcrA. This work showed the refined procedures of the antibiotics binding to CcrA and might provide useful information hint for the new strategy of developing the novel and innovative super-bacterial antibiotics.

Screening and identification of bioactive components resistant to metallo-beta-lactamase from Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography.

The screening and identification of bioactive components, which are effectively resistant to metallo-beta-lactamase (MßL), were studied in the alcohol extract of Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography. Taking bizinc metalloenzyme beta-lactamase II from Bacillus cereus (Bc II) and monozinc metalloenzyme CphA from aeromonas hydrophila (CphA) as examples, we studied the feasibility of this scheme based on the construction of metalloenzyme-immobilized chromatographic model. It was found that the Bc II- and CphA-immobilized chromatographic column could be used not only to explore the interaction between the MßL and their specific ligands, but also to screen the bioactive components from traditional Chinese medicine. The Bc II-and CphA-immobilized columns were used to screen the bioactive components from the alcohol extract of Schisandra chinensis (Turcz.) Baill. Time-of-flight tandem mass spectrometry analysis and molecular docking revealed that isobutyl 3-O-sulfo-ß-D-galactopyranoside is the effective bioactive components that could bind with metalloenzyme Bc II. It is believed that our current work may provide a methodological reference for screening MßL inhibitors from traditional Chinese medicine.

An illustrated key to the species of subgenus Gyrostoma Kirby, 1828 (Hymenoptera, Vespidae, Polistinae) from China, with discovery of Polistes (Gyrostoma) tenuispunctia Kim, 2001.

The Chinese species of the subgenus Gyrostoma Kirby, 1828 of the polistine genus Polistes are reviewed. An illustrated key to the seven species of the subgenus known from China is given. New synonymy are proposed for Polistes rothneyi Cameron, 1900 =P. rothneyi grahami van der Vecht, 1968, syn. nov.; =P. r. hainanensis van der Vecht, 1968, syn. nov.; =P. r. iwatai van der Vecht, 1968, syn. nov.; =P. r. gressitti van der Vecht, 1968, syn. nov.; =P. r. tibetanus van der Vecht, 1968, syn. nov.; = P. r. yayeyamae Matsumura, 1908, syn. nov.; =P. r. koreanus van der Vecht, 1968, syn. nov.; =P. r. sikkimensis van der Vecht, 1968, syn. nov.. P. (Gyrostoma) tenuispuntia Kim, 2001 is newly recorded from China. Its nest is described for the first time. Compared with the sympatric and similar species P. rothneyi f. grahami the nest is concealed in a hollow space instead of having the nest directly exposed under an eave as in P. rothneyi. The differences in nest architecture are briefly discussed.