Icariin improves oxidative stress injury during ischemic stroke via inhibiting mPTP opening.

BACKGROUND: Ischemic stroke presents a significant threat to human health due to its high disability rate and mortality. Currently, the clinical treatment drug, rt-PA, has a narrow therapeutic window and carries a high risk of bleeding. There is an urgent need to find new effective therapeutic drugs for ischemic stroke. Icariin (ICA), a key ingredient in the traditional Chinese medicine Epimedium, undergoes metabolism in vivo to produce Icaritin (ICT). While ICA has been reported to inhibit neuronal apoptosis after cerebral ischemia-reperfusion (I/R), yet its underlying mechanism remains unclear. METHODS: PC-12 cells were treated with 200 µM H2O2 for 8 h to establish a vitro model of oxidative damage. After administration of ICT, cell viability was detected by Thiazolyl blue tetrazolium Bromide (MTT) assay, reactive oxygen species (ROS) and apoptosis level, mPTP status and mitochondrial membrane potential (MMP) were detected by flow cytometry and immunofluorescence. Apoptosis and mitochondrial permeability transition pore (mPTP) related proteins were assessed by Western blotting. Middle cerebral artery occlusion (MCAO) model was used to establish I/R injury in vivo. After the treatment of ICA, the neurological function was scored by ZeaLonga socres; the infarct volume was observed by 2,3,5-Triphenyltetrazolium chloride (TTC) staining; HE and Nissl staining were used to detect the pathological state of the ischemic cortex; the expression changes of mPTP and apoptosis related proteins were detected by Western blotting. RESULTS: In vitro: ICT effectively improved H2O2-induced oxidative injury through decreasing the ROS level, inhibiting mPTP opening and apoptosis. In addition, the protective effects of ICT were not enhanced when it was co-treated with mPTP inhibitor Cyclosporin A (CsA), but reversed when combined with mPTP activator Lonidamine (LND). In vivo: Rats after MCAO shown cortical infarct volume of 32-40%, severe neurological impairment, while mPTP opening and apoptosis were obviously increased. Those damage caused was improved by the administration of ICA and CsA. CONCLUSIONS: ICA improves cerebral ischemia-reperfusion injury by inhibiting mPTP opening, making it a potential candidate drug for the treatment of ischemic stroke.

A radical 1,4-aryl migration enables nickel-catalysed remote cross-electrophile coupling of ß-bromo amino acid esters with vinyl triflates.

A radical 1,4-aryl migration enabling a cross-electrophile coupling reaction toward remote transalkylation of N-benzyl alanine has been developed. In this strategy, with the occurrence of a radical-mediated Turce-Smiles rearrangement, key α-aminoalkyl radicals are generated. The as-formed α-aminoalkyl radical serves as a robust coupling partner for cross-electrophilic coupling with vinyl triflates, affording a series of olefin-tethered amino acid motifs.

Nickel-Catalyzed Radical Heck-Type C(sp3)-C(sp2) Coupling Cascades Enabled by Bromoalkane-Directed 1,4-Aryl Shift: Access to Olefinated Arylalanines.

A bromoalkane-directed radical 1,4-aryl shift strategy for nickel-catalyzed reductive Heck-type C(sp3)-C(sp2) coupling cascades of α-amino-ß-bromocarboxylic acid esters with α-trifluoromethyl alkenes for producing gem-difluorinated arylalanines is presented. The α-aminoalkyl radicals generated from neophyl-type aryl migration function as robust coupling partners to allow for further Giese-type addition with electron-deficient α-trifluoromethyl alkenes and vinyl sulfones, thereby realizing a new radical cascade for the simultaneous installation of an aromatic ring and olefin motif into amino acid backbones.

Antimicrobial Properties of Chitosan and Chitosan Derivatives in the Treatment of Enteric Infections.

Antibiotics played an important role in controlling the development of enteric infection. However, the emergence of antibiotic resistance and gut dysbiosis led to a growing interest in the use of natural antimicrobial agents as alternatives for therapy and disinfection. Chitosan is a nontoxic natural antimicrobial polymer and is approved by GRAS (Generally Recognized as Safe by the United States Food and Drug Administration). Chitosan and chitosan derivatives can kill microbes by neutralizing negative charges on the microbial surface. Besides, chemical modifications give chitosan derivatives better water solubility and antimicrobial property. This review gives an overview of the preparation of chitosan, its derivatives, and the conjugates with other polymers and nanoparticles with better antimicrobial properties, explains the direct and indirect mechanisms of action of chitosan, and summarizes current treatment for enteric infections as well as the role of chitosan and chitosan derivatives in the antimicrobial agents in enteric infections. Finally, we suggested future directions for further research to improve the treatment of enteric infections and to develop more useful chitosan derivatives and conjugates.

Effects from metal ion in tumor endothelial marker 8 and anthrax protective antigen: BioLayer Interferometry experiment and molecular dynamics simulation study.

One of the anthrax receptors, tumor endothelial marker 8 (TEM8), is reported to be a potential anticancer target due to its over-expression during tumor angiogenesis. To extend our BioLayer Interferometry study in PA-TEM8 binding, we present a computational approach to reveal the role of an integral metal ion on receptor structure and binding thermodynamics. We estimated the interaction energy between PA and TEM8 using computer simulation. Consistent with experimental study, computational results indicate the metal ion in TEM8 contributes significantly to the binding affinity, and PA-TEM8 binding is more favorable in the presence of Mg2+ than Ca2+ . Further, computational analysis suggests that the differences in PA-TEM8 binding affinity are comparable to the closely related integrin proteins. The conformation change, which linked to changes in activity of integrins, was not found in TEM8. In the present of Mg2+ , TEM8 remains in a conformation analogous to an integrin open (high-affinity) conformation. © 2017 Wiley Periodicals, Inc.

Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily.

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs.

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed.

Specificity and catalytic mechanism in family 5 uracil DNA glycosylase.

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.