Ying Mu

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 Uu2009μM−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.

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.

Single Chain Antibody Displays Glutathione S-Transferase Activity *

Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat θ-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.

An insight into the protection of rat liver against ischemia/reperfusion injury by 2‐selenium‐bridged β‐cyclodextrin

Aim:u2002 The reperfusion following liver ischemia results in the damage and apoptosis of hepatocytes. The aim of this study was to investigate the possible effects and mechanism of a new synthesized glutathione peroxidase (GPX) mimic, 2‐selenium‐bridged β‐cyclodextrin (2‐SeCD), on rat liver ischemia‐reperfusion (I/R) injury.

Protection of epidermal cells against UVB injury by the antioxidant selenium-containing single-chain Fv catalytic antibody

The antioxidant effect of selenium-containing single-chain Fv catalytic antibody (Se-scFv2F3), a new mimic of glutathione peroxidase, was confirmed using a model system in which cultured rat skin epidermal cells were injured by ultraviolet B (UVB). The cell damage was characterized in terms of lipid peroxidation of the cells, cell viability, and cell membrane integrity. The injury effects of UVB and protection effects of Se-scFv2F3 on the cells were studied using the model system. UVB can damage the cells severely. Upon precultivation of the cells with 0.4U/ml Se-scFv2F3, however, the damage was significantly reduced as shown by the increase in cell viability, the decrease in the malondialdehyde and hydrogen peroxide levels, and the normalization of lactate dehydrogenase activity. In addition, a novel finding that Se-scFv2F3 can stimulate cultured epidermal cells to proliferate under certain conditions was observed.

The Small Glutathione Peroxidase Mimic 5P May Represent a New Strategy for the Treatment of Liver Cancer

Glutathione peroxidase (GPx) is an antioxidant protein containing selenium. Owing to the limitations of native GPx, considerable efforts have been made to develop GPx mimics. Here, a short 5-mer peptides (5P) was synthesized and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Enzyme coupled assays were used to evaluate GPx activity. The cell viability and apoptosis of H22 cells were tested, and mice bearing H22 cell-derived tumors were used to determine the effects of 5P on tumor inhibition. In comparison with other enzyme models, 5P provided a suitable substrate with proper catalytic site positions, resulting in enhanced catalytic activity. In our mouse model, 5P showed excellent inhibition of tumor growth and improved immunity. In summary, our findings demonstrated the design and synthesis of the small 5P molecule, which inhibited tumor growth and improved immunity. Notably, 5P could inhibit tumor growth without affecting normal growth. Based on these advantages, the novel mimic may have several clinical applications.

20(S)-Ginsenoside Rh2 Induce the Apoptosis and Autophagy in U937 and K562 Cells

Acute myeloid leukemia (AML) and Chronic myelogenous leukemia (CML) are common leukemia in adults. 20(S)-GRh2 is an important bioactive substance that is present in Panax ginseng. However, there are no investigations that deal with the comparison of apoptosis, the occurrence of autophagy, and the relationship between apoptosis and autophagy after being treated with 20(S)-GRh2 in AML and CML. In this study, we explored the effect of 20(S)-GRh2 on the AML and CML (U937 and K562). Fluorescence microscopy, CCK-8, Quantitative realtime PCR, Western blot, transmission electron microscopy (TEM), and flow cytometric analysis were used to detect the occurrence of cell proliferation inhibition, apoptosis, and autophagy. By using the above methods, it was determined that apoptosis induced by 20(S)-GRh2 was more obvious in K562 than U937 cells and 20(S)-GRh2 could generate autophagy in K562 and U937 cells. When pretreated by a specific inhibitor of autophagy, (3-methyladenine), the 20(S)-GRh2-induced apoptosis was enhanced, which indicated that 20(S)-GRh2-induced autophagy may protect U937 and K562 cells from undergoing apoptotic cell death. On the other hand, pretreated by an apoptosis suppressor (Z-VAD-FMK), it greatly induced the autophagy and partially prevented 20(S)-GRh2 induced apoptosis. This phenomenon indicated that 20(S)-GRh2-induced autophagy may serve as a survival mechanism and apoptosis and autophagy could act as partners to induce cell death in a cooperative manner. These findings may provide a rationale for future clinical application by using 20(S)-GRh2 combined autophagy inhibitors for AML and CML.

Study on the Correlation between Gene Expression and Enzyme Activity of Seven Key Enzymes and Ginsenoside Content in Ginseng in Over Time in Ji’an, China

Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji’an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.