Scavenging ROS: Superoxide Dismutase/Catalase Mimetics by the Use of an Oxidation-Sensitive Nanocarrier/Enzyme Conjugate

Reactive Oxygen Species (ROS) are quintessential inflammatory compounds with oxidizing behavior. We have successfully developed a micellar system with responsiveness at the same time to two of the most important ROS: superoxide and hydrogen peroxide. This allows for an effective and selective capture of the two compounds and, in perspective, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity toward superoxide with that of hydrophobic thioethers toward hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then “mopped-up” by the polysulfides: this <i>modus operandi</i> is functionally analogous to the SOD/catalase combination, with the advantages of (a) being based on a single and more stable system, and (b) a higher overall efficiency due the physical proximity of the two ROS-reactive centers (SOD and polysulfides)

TM domain separation is required for cell spreading.

<p>(A) Stably transfected cells were seeded on immobilized Fbg (20μg/mL) with or without 1mM DTT for 30min at 37°C and visualized using differential interference contrast (DIC). Images are representatives from one of three independent assays. White bar: 10μm. (B) Average areas of adherent cells were quantified by pixel. The error bars represent S.D. from 100 randomly chosen cells.</p

All α_IIbβ₃ mutants were successfully expressed with high disulfide-bridge formation efficiency.

<p>(A)Integrin α_IIbβ₃ constructs used in our study. B1: Wild type integrin α_IIbβ₃; B2: Disulfide-bonded α_IIbβ₃(α_IIb_W968C, β₃_I693C); B3: α truncated α_IIbβ₃(α_IIb1-990); B4: α truncated, disulfide-bonded α_IIbβ₃ (α_IIb1-990_W968C, β₃_I693C). (B) Cell lines stably expressing WT and mutated integrin α_IIbβ₃ as evaluated by three different monoclonal antibodies. B1 (WT α_IIbβ₃), B2 (disulfide-bonded α_IIbβ₃ mutant), B3 (α-truncated α_IIbβ₃), and B4 (disulfide-bonded α-truncated α_IIbβ₃). The solid line and dashed line represent untransfected CHO-K1 cells and stable transfectants, respectively. AP3, 7E3, and 10E5 are mAbs targeting different domains of the α_IIbβ₃ heterodimer. (C) Disulfide bonds formed between α and β subunits with high efficiency. Cells labeled with S³⁵ were lysed and subjected to immunoprecipitation by anti-α_IIb mAb 10E5. Immunoprecipitated protein was then resolved by SDS-PAGE and visualized by radioautography. 2mM DTT was used to reduce the disulfide bridge in TM-clasped mutants.</p

TM domain separation but not α_IIb cytoplasmic domain is required for recruitment and phosphorylation of paxillin.

<p>(A) Recruitment of phosphorylated paxillin (PaxpY31, green) to FA sites (blue) was observed in both wild type (B1) and α_IIb-truncated mutant (B3) but not in the two disulfide-bonded mutants (B2 and B4). Note that recruitment of phosphorylated paxillin was normal in the α_IIb-truncated mutant indicating a dispensable role of the α_IIb cytoplasmic domain in paxillin recruitment. (B) Addition of 1mM DTT restored recruitment of phosphorylated paxillin in both of the disulfide-bonded mutants. White bar: 10μm.</p

TM separation promotes activation of FAK whereas PI3K regulates Akt and Erk1/2 but not FAK in outside-in signaling.

<p>Cells were seeded on Fbg (20μg/mL)-coated dishes with or without 1mM DTT or with 1.5μM/mL Wortmannin at 37°C for 1h and then lysed and subjected to western blot. Cells seeded on 1% BSA coated dishes were used as control. (A)FAK activation induced by immobilized ligand Fbg is TM domain separation dependent. TM-clasped mutants expressed reduced phosphorylation on Y397. (B) Treatment with Wortmannin ablated Akt and Erk1/2 activation. Removing Wortmannin before cell spreading restored Erk1/2 activation without inducing Akt activation. This may implicate that PI3K but not Akt activity is required for cell survival and proliferation mediated by Erk1/2 in outside-in signaling. (C) Wortmannin treatment did not alter activation of FAK, implicating parallel pathways in outside-in signaling.</p

Inhibition of PI3K and Src did not affect cell spreading, FAK/paxillin recruitment, or phosphorylation.

<p>(A, B) FAK and paxillin were phosphorylated and recruited to FAs normally in WT (B1) and the α_IIb-truncated mutant (B3) with control treatment (DMSO). TM clasping abolished the events as previously observed; (C, D) Treatment with 1.5μM/mL Wortmannin did not affect recruitment of phosphorylated FAK and paxillin. (E, F) PP1 (15μM/mL) had little to no effect on cell spreading with regard to FAK/paxillin phosphorylation and recruitment. White bar: 10μm.</p

TM domain disassociation is required for cytoplasmic domain dissociation-induced high affinity integrin as measured by soluble ligand binding.

<p>Binding of ligand mimetic mAb PAC-1(A) and fibrinogen (Fbg, B) to B1 through B4 cells in the presence of either EDTA (5mM), Ca²⁺ (5mM), Mn²⁺ (1mM), or Ca²⁺(5mM) with DTT (4mM) as indicated. Error bars represent standard deviation (S.D.) from three independent assays.</p

TM domain separation is required for FA formation, actin fiber organization, FAK activation and recruitment to FA sites in outside-in signaling.

<p>(A) Clasping of TM domains ablated FA formation and disrupted actin filaments organization. Note that α truncation led to an even distribution of FAs around adherent cells. (B) Treatment with 1mM DTT largely restored cell spreading and FA assembly. Green: focal adhesions (labeled with anti-vinculin antibody); Red: actin filaments (labeled with TRITC-conjugated Phalloidin). White bar: 10μm. (C) Activated FAK (FAKpY397) was recruited normally to FAs in WT (B1) and the α-truncated mutant (B3), but recruitment was abolished by clasping the TM domains (B2 and B4). (D) Treatment with 1mM DTT restored recruitment of phosphorylated FAK to FAs. Green: FAKpY397; Red: actin filaments; Blue: FA marker vinculin. White bar: 10μm.</p

Syntheses, Characterization, and Ethylene Polymerization of Half-Sandwich Zirconium Complexes with Tridentate Imino−Quinolinol Ligands

A series of half-sandwich zirconium complexes with imino-quinolinol ligands have been synthesized and characterized. The catalytic behaviors of these complexes toward ethylene polymerization were investigated in the presence of methylaluminoxane (MAO) as a cocatalyst. The catalytic behaviors were highly affected by the substituent in both cyclopentadienyl and imino−quinolinol ligands. The Cp analogue complexes CpZr[ONNR]Cl2 (1a−e) exhibited high activities up to 1.34 × 107 g of PE (mol of Zr)−1 h−1, whereas the Cp* analogue complexes Cp*Zr[ONNR]Cl2 (2a−e) also showed moderate activities for ethylene polymerization

Syntheses, Characterization, and Ethylene Polymerization of Half-Sandwich Zirconium Complexes with Tridentate Imino−Quinolinol Ligands

A series of half-sandwich zirconium complexes with imino-quinolinol ligands have been synthesized and characterized. The catalytic behaviors of these complexes toward ethylene polymerization were investigated in the presence of methylaluminoxane (MAO) as a cocatalyst. The catalytic behaviors were highly affected by the substituent in both cyclopentadienyl and imino−quinolinol ligands. The Cp analogue complexes CpZr[ONNR]Cl2 (1a−e) exhibited high activities up to 1.34 × 107 g of PE (mol of Zr)−1 h−1, whereas the Cp* analogue complexes Cp*Zr[ONNR]Cl2 (2a−e) also showed moderate activities for ethylene polymerization