Guimin Luo

Determination of dynamic interfacial tension and its effect on droplet formation in the T-shaped microdispersion process.

Interfacial tension is an important physical property affecting the droplet formation process in microfluidic devices. This work presents the variation of dynamic interfacial tension caused by slow adsorption of surfactant, as well as its influence on the liquid/liquid microdispersion process in a T-shaped microchannel. Using hexane/water-Tween 20 as the working system, it was observed that the droplet size changed with the variation of surfactant concentration when the concentration of Tween 20 was lower than 10 mmol/L, but hardly changed at higher concentrations, which was caused by the unsaturated adsorption and saturated adsorption of surfactant, respectively. The saturated interfacial tension was measured with an interfacial tension meter, and the relationship between the interfacial tension and the droplet diameter was established. Accordingly, the dynamic interfacial tension with unsaturated adsorption of surfactant was determined. The main factors affecting the dynamic interfacial tension were discussed, and a semiempirical equation was established to characterize those effects.

A novel cyclodextrin-derived tellurium compound with glutathione peroxidase activity.

A novel dicyclodextrinyl ditelluride (2‐TeCD) compound was devised as a functional mimic of the glutathione peroxidase (GPX) enzymes that normally remove hydroperoxides from the cell. The GPX activity of the mimic was found to be 46.7 U μM−1, which is 46 times as active as Ebselen, a well‐known GPX mimic. A detailed steady‐state kinetic study was undertaken to probe the reason for the high catalytic efficiency of 2‐TeCD. This high efficiency can be explained based on both the binding of the substrate to the cyclodextrin and the catalytic mechanism of 2‐TeCD, which is different from that of diselenide compounds. 2‐TeCD exhibits good water solubility and is chemically and biologically stable. The biological effect of 2‐TeCD was evaluated by its ability to protect mitochondria from oxidative damage. 2‐TeCD exhibited excellent antioxidant capacity in comparison with Ebselen.

A Semisynthetic Glutathione Peroxidase with High Catalytic Efficiency: Selenoglutathione Transferase

Glutathione peroxidase (GPX) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). GPX therefore has potential therapeutic value as an antioxidant, but its pharmacological development has been limited because GPX uses a selenocysteine as its catalytic group and it is difficult to generate selenium-containing proteins with traditional recombinant DNA technology. Here, we show that naturally occurring proteins can be modified to generate GPX activity. The rat theta-class glutathione transferase T2-2 (rGST T2-2) presents an ideal scaffold for the design of a novel GPX catalyst because it already binds GSH and contains a serine close to the substrate binding site, which can be chemically modified to bind selenium. The modified Se-rGST T2-2 efficiently catalyzes the reduction of hydrogen peroxide, and the GPX activity surpasses the activities of some natural GPXs.

Incorporation of Tellurocysteine into Glutathione Transferase Generates High Glutathione Peroxidase Efficiency

A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.

A novel dicyclodextrinyl ditelluride compound with antioxidant activity

Reactive oxygen species (ROS) primarily arise from products of normal metabolic activities and are thought to be the etiology of many diseases. A novel dicyclodextrinyl ditelluride (2‐TeCD) compound was designed to be a functional mimic of the glutathione peroxidase that normally removes ROS. 2‐TeCD exhibited highly catalytic efficiency and good water solubility. Antioxidant activity was studied by using ferrous sulfate/ascorbate‐induced mitochondria damage model system. 2‐TeCD protected the mitochondria against oxidative damage in a dose‐dependent manner and exhibited also great antioxidant ability in comparison with 2‐phenyl‐1,2‐benziososelenazol‐3(2H)‐one. The mimic may result in better clinical therapies for the treatment of ROS‐mediated diseases.

Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency.

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibodys binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240-1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251-255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5-10%, 15-20% and 25-30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8 M urea by Co(2+)-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46 x 10(5) M(-1) and 1.03 x 10(5) M(-1) respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394 units/micromol, which approaches the activity of rabbit liver GPX.

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.

Selenium-containing 15-Mer Peptides with High Glutathione Peroxidase-like Activity

Glutathione peroxidase (GPX) is one of the most crucial antioxidant enzymes in a variety of organisms. Here we described a new strategy for generating a novel GPX mimic by combination of a phage-displayed random 15-mer peptide library followed by computer-aided rational design and chemical mutation. The novel GPX mimic is a homodimer consisting of a 15-mer selenopeptide with an appropriate catalytic center, a specific binding site for substrates, and high catalytic efficiency. Its steady state kinetics was also studied, and the values of kcat/KmGSH and kcat/ KmH2O2 were found to be similar to that of native GPX and the highest among the existing GPX mimics. Moreover, the novel GPX mimic was confirmed to have a strong antioxidant ability to inhibit lipid peroxidation by measuring the content of malondialdehyde, cell viability, and lactate dehydrogenase activity. Importantly, the novel GPX mimic can penetrate into the cell membrane because of its small molecular size. These characteristics endue the novel mimic with potential perspective for pharmaceutical applications.

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity

A 6A,6A′‐dicyclohexylamine‐6B,6B′‐diselenide‐bis‐β‐cyclodextrin (6‐CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, β‐cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6‐CySeCD exhibits better GPX activity than 6,6′‐diselenide‐bis‐cyclodextrin (6‐SeCD) and 2‐phenyl‐1,2‐benzoisoselenazol‐3(2H)‐one (Ebselen) in the reduction of H2O2, tert‐butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping‐pong mechanism was observed in steady‐state kinetic studies on 6‐CySeCD‐catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate‐binding site should match the size and shape of the substrate and second, incorporation of an imido‐group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6‐SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate‐induced mitochondria damage system, and this implies its prospective therapeutic application.

2-Tellurium-bridged β-cyclodextrin, a thioredoxin reductase inhibitor, sensitizes human breast cancer cells to TRAIL-induced apoptosis through DR5 induction and NF-κB suppression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumor activity via membrane receptors on cancer cells without deleterious side effects for normal tissue. Unfortunately, breast cancer cells, as many other cancer types, develop resistance to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. 2-Tellurium-bridged β-cyclodextrin (2-TeCD) is a synthetic organotellurium compound, with both glutathione peroxidase-like catalytic ability and thioredoxin reductase inhibitor activity. In the present study, we reported that 2-TeCD sensitized TRAIL-resistant human breast cancer cells and xenograft tumors to undergo apoptosis. In vitro, 2-TeCD efficiently sensitized MDA-MB-468 and T47D cells, but not untransformed human mammary epithelial cells, to TRAIL-mediated apoptosis, as evidenced by enhanced caspase activity and poly (adenosine diphosphate-ribose) polymerase cleavage. From a mechanistic standpoint, we showed that 2-TeCD treatment of breast cancer cells significantly upregulated the messenger RNA and protein levels of TRAIL receptor, death receptor (DR) 5, in a transcription factor Sp1-dependent manner. 2-TeCD treatment also suppressed TRAIL-induced nuclear factor-κB (NF-κB) prosurvival pathways by preventing cytosolic IκBα degradation, as well as p65 nuclear translocation. Consequently, the combined administration suppressed anti-apoptotic molecules that are transcriptionally regulated by NF-κB. In vivo, 2-TeCD and TRAIL were well tolerated in mice and their combination significantly inhibited growth of MDA-MB-468 xenografts and promoted apoptosis. Upregulation of DR5 and downregulation of NF-κB by the dual treatment were also observed in tumor tissues. Overall, 2-TeCD sensitizes resistant breast cancer cells to TRAIL-based apoptosis in vitro and in vivo. These findings provide strong evidence for the therapeutic potential of this combination against breast cancers.