An insight into the protection of rat liver against ischemia/reperfusion injury by 2-selenium-bridged beta-cyclodextrin.

AIM: 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 beta-cyclodextrin (2-SeCD), on rat liver ischemia-reperfusion (I/R) injury. METHODS: Male Wistar rats (n = 32) were randomly divided into four groups: I. sham-operated group, II. I/R group, III. I/R +2-SeCD group, IV. I/R + Ebselen group. Hepatic I/R was administered by 90 min of ischemia and 12 h of reperfusion. Liver tissues were collected at the end of reperfusion period for measurement of various biochemical parameters. RESULTS: The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and tissue malondialdehyde, myeloperoxidase levels were increased in I/R group, while the increase was significantly reduced by 2-SeCD treatment. The glutathione level, depressed by I/R, was elevated back to normal levels by treatment with 2-SeCD. Severe hepatic damage were observed by light and transmission electron microscopy whilst pretreatment with 2-SeCD resulted in tissue and cellular preservation. Furthermore, 2-SeCD reduced cytochrome c release from mitochondria and subsequent DNA fragmentation by regulating Bcl-2/Bax expression ratio. RESULTS suggested that 2-SeCD was more effective than ebselen in the reversal of the alteration in tissue structural and biochemical parameters caused by I/R injury. CONCLUSION: 2-selenium-bridged beta-cyclodextrin playes an important role in the protection of liver against I/R injury and this treatment may be a novel pharmacological agent for liver surgery.

The effect of a cholesterol liquid crystalline structure on osteoblast cell behavior.

To investigate the effect of a liquid crystalline structure on cell behavior, polymethylsiloxane-graft-(10-cholesteryloxydecanol) was specially designed to get a thermotropic liquid crystalline polymer. Results of Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel permeation chromatography (GPC) indicated that cholesterol was successfully covalently grafted onto polymethylhydrosiloxane via decamethylene 'flexible spacer'. Differential scanning calorimetry (DSC) and polarized optical microscopy (POM) investigations revealed that the copolymer with 44.9% mesogenic unit showed obvious thermotropic liquid crystalline transition at about 124.9 degrees C. Polymer films were prepared by spin coating on clean glass plates from 5 mg ml(-1) toluene solutions of the copolymers. The POM investigation indicated that while the unannealed films (SC15, SC45) showed no liquid crystalline structure, the films which were annealed in vacuo at 140 degrees C for 9 h and then quenched to room temperature (SC15C, SC45C) formed discrete island-like liquid crystalline and continuous liquid crystalline structures, respectively. Osteoblast cells (MC3T3) were chosen to test the cell behavior of annealed and unannealed films. In comparison to unannealed films, the annealed films with a cholesterol liquid crystalline structure could promote osteoblast cell attachment and growth significantly.

A novel biomimetic polymer as amphiphilic surfactant for soluble and biocompatible carbon nanotubes (CNTs).

Novel amphiphilic diblock copolymer, cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (CPMPC), which has poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as hydrophilic segment and cholesterol as hydrophobic segment, was specially designed as amphiphilic surfactant to achieve water-soluble and biocompatible carbon nanotubes (CNTs). The pristine CNTs were facilely dispersed via non-covalently binding the zwitterionic phosphorylcholine-based amphiphile onto the surfaces of the CNTs. It is interesting to find that CPMPC shows better CNTs solubilizing ability compared with the surfactant of pyrene-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (PPMPC). The biocompatibility of the CPMPC stabilized CNTs was evaluated using cholesterol-end-capped poly(2-(dimethylamino) ethyl methacrylate) (CPDMAEMA), cholesterol-end-capped poly(acrylic acid) (CPAA) and cholesterol-end-capped poly(ethylene oxide) (CPEG) as surfactants for CNTs as controls. While CPDMAEMA stabilized CNTs and CPAA stabilized CNTs showed obvious cytotoxicity, cytotoxicity of this novel zwitterionic phosphorylcholine-based amphiphile stabilized CNTs was not observed as indicated by cell culture. The biocompatible CNTs represent an excellent nano-object for potential biomedical applications.

Zwitterionic phosphorylcholine as a better ligand for stabilizing large biocompatible gold nanoparticles.

Zwitterionic phosphorylcholine showed better stabilization than oligo(ethylene glycol) in protecting big gold nanoparticles.

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.

Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein.

For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity.

A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-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 H(2)O(2), 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.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

[Nanobiomaterials].

Nanobiomaterial is a multidisciplinary scientific field with roots in life science, material science and nanotechnology. The basic and application researches of nanobiomaterials have been the hot topic in the materials research for biomedicine and biotechnology, which have developed quickly in biomedical implant and intervention medicine, tissue engineering and regenerative medicine, and drug/gene delivery system. This review focuses on the potential of nanobiomaterials including biocompatible surface, tissue engineering and regenerative materials, new drug/gene delivery system and bioanalysis system, with an attempt to explore their possible applications in clinical practice.

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-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.

Cyclodextrin-derived mimic of glutathione peroxidase exhibiting enzymatic specificity and high catalytic efficiency.

To elucidate the relationships between molecular recognition and catalytic ability, we chose three assay systems using three different thiol substrates, glutathione (GSH), 3-carboxyl-4-nitrobenzenethiol (CNBSH), and 4-nitrobenzenethiol (NBSH), to investigate the glutathione peroxidase (GPx) activities of 2,2'-ditellurobis(2-deoxy-beta-cyclodextrin) (2-TeCD) in the presence of a variety of structurally distinct hydroperoxides (ROOH), H2O2, tert-butyl peroxide (tBuOOH), and cumene peroxide (CuOOH), as the oxidative reagent. A comparative study of the three assay systems revealed that the cyclodextrin moiety of the GPx mimic 2-TeCD endows the molecule with selectivity for ROOH and thiol substrates, and hydrophobic interactions are the most important driving forces in 2-TeCD complexation. Furthermore, in the novel NBSH assay system, 2-TeCD can catalyze the reduction of ROOH about 3.4 x 10(5) times more efficiently than diphenyl diselenide (PhSeSePh), and its second-order rate constants for thiol are similar to some of those of native GPx. This comparative study confirms that efficient binding of the substrate is essential for the catalytic ability of the GPx mimic, and that NBSH is the preferred thiol substrate of 2-TeCD among the chosen thiol substrates. Importantly, the proposed mode of action of 2-TeCD imitates the role played by several possible noncovalent interactions between enzymes and substrates in influencing catalysis and binding.

Novel biomimetic polymersomes as polymer therapeutics for drug delivery.

Novel amphiphilic diblock copolymers, cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (CMPC), which have poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) as hydrophilic segment and cholesterol as hydrophobic segment, was specially designed as drug delivery systems. Fluorescence probe technique and transmission electron microscope (TEM) characterizations indicated that this novel amphiphilic copolymer formed micelles structure in water and the critical micelle concentration (CMC) was determined to be 1.57 x 10(-7) mol/l. A commercial obtained polymeric amphiphiles, Cholesterol end capped PEO (CPEO), which had a similar structure with CMPC, was used as a control in the cytotoxicity test. While CPEO showed obvious cytotoxicity, cytotoxicity of this novel amphiphiles was not observed as indicated by cell culture. Anti-cancer drug adriamycin (ADR) was incorporated into the micelles by oil-in-water method. The size of the drug-containing micelles was less than 200 nm, and the size distribution of the drug-containing micelles showed a narrow and monodisperse unimodal pattern. The release rate of ADR from the nanosphere was slow and the release continued over 7 days and the release rate decreased with the increase of molecular weights of the copolymer and the amount of the drug entrapped. These experimental results suggested that the nanoparticles prepared from CMPC block copolymers could be a good candidate for injectable drug delivery carrier.

Fabrication of thromboresistant multilayer thin film on plasma treated poly (vinyl chloride) surface.

Layer-by-layer deposited anticoagulant multilayer films were prepared on ammonia plasma treated poly (vinyl chloride) (PVC). Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) and contact angle results revealed the presence of -NH2 on the ammonia plasma treated PVC surfaces and the layer-by-layer self-assembly process. The stability of multilayer film was studied with the radio labeled method. The remainder bovine serum albumin (BSA) in cross-linked 5(heparin/BSA) multilayer films dipped in phosphate buffered saline (PBS, pH 7.4) was more than 90% in 40 days. The static platelet adhesion result indicated the anticoagulant multilayer films deposited on the plasma treated PVC reduced platelet adhesion drastically and no thrombus forming. The plasma recalcification time revealed that the multilayer modified surfaces greatly prolonged the plasma recalcification time. Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices.

The effect of formation of the liquid crystalline phase on the blood compatibility of a cholesterol modified silicone.

Cholesterol modified silicones were synthesized by grafting copolymerization of 10-Cholesteryloxydecanol onto polymethylhydrosiloxane (PMHS). Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance ((1)H-NMR) spectroscopy and gel permeation chromatography (GPC) confirmed the chemical structures of polymers. Differential scanning calorimetry (DSC) and polarized optical microscopy (POM) results indicated the mesogenic properties of those polymers. The modified silicone with 45% 10-Cholesteryloxydecanyl (SC45) indicated obvious thermotropic liquid crystalline transform at about 122-124.9 degrees C. The thermotropic liquid crystalline phase could be retained at room temperature via a special annealing/quenching process. The anneal-quenched film (SC45C) formed continuous liquid crystalline phase, whereas the unannealed films presented amorphous structure. The blood compatibility of the coatings was assessed from SEM observation of the platelet's adhesion to coating surface and plasma recalcification time (PRT). The results revealed that the formation of the liquid crystalline phase could greatly improve the in vitro blood compatibility of the materials. The positive results of liquid crystalline onto haemocompatibility allow broad potential in biomaterials.

Novel biomimetic surfactant: synthesis and micellar characteristics.

Novel biomimetic surfactants based on cholesterol as the hydrophobic segment and poly[2-(methacryloyloxy)ethyl phosphorylcholine] (pMPC) as the hydrophilic segment were synthesized in the present study by atom transfer radical polymerization (ATRP) of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) using a cholesterol-based macroinitiator. The association behavior of cholesterol-block-poly[2-(methacryloyloxy)ethyl phosphorylcholine] (Chol-pMPCs) in aqueous solution was studied by (1)H NMR spectroscopy, fluorescence probe technique, and atomic force microscopy (AFM). The (1)H NMR spectrum of the polymer in CD(3)OD showed both the cholesterol group and the phosphorylcholine group while the cholesterol group did not appeared in the (1)H NMR spectrum of the polymer in D(2)O, which implied the formation of a micelle structure. Fluorescence excitation spectra of a pyrene probe solubilized in the aggregates of Chol-pMPCs suggested the presence of a critical micelle concentration (cmc) in water. The critical micelle concentrations of the polymers CMPC10, CMPC20 and CMPC40 were determined to be 7.27 x 10(-3), 13.47 x 10(-3), and 20.77 x 10(-3) mg . mL(-1), respectively. AFM images of the aggregates on mica suggested that the pMPC block formed the biocompatible micelle coronas and the cholesterol block formed the hydrophobic micelle cores. These new biomimetic diblock copolymers were evaluated as "stealthy" nanocapsules for the delivery of hydrophobic drugs. The anti-cancer drug adriamycin (ADR) was chosen as a hydrophobic drug to be incorporated into the inner core of the micelles and the morphology of the drug-loaded micelles were observed by AFM.

Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system.

Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.

A novel crosslinkable polymer as drug-loaded coating for biomedical device.

A novel copolymer has been synthesized by the radical polymerization of poly (ethylene oxide) methacrylate, stearyl methacrylate, hydroxypropyl methacrylate and trimethoxysilylpropyl methacrylate. The polymer was characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel permeation chromatography. The crosslinkable coating was prepared by dip-coating 5mg/ml solution in tetrahydrofuran onto glass substrate. A stable crosslinked coating was obtained after curing the coating at 70 degrees C for 9 h. Contact angle results indicated the possible reorganization of the surface amphiphilic molecule which interpreted the excellent biocompatibility revealed by the results of the platelet adhesion and plasma recalcification time. Rhodamine S and Cibacron Blue were used as model drugs to prepare drug-containing coating at the same conditions. Drug-releasing curves indicated that the mechanism of the release is approximately Fickian release.

Water-dispersed bone morphogenetic protein nanospheres prepared by co-precipitation method.

A modified complex coacervation-co-precipitation method was used to prepare bone morphogenetic protein (BMP)-loaded nanospheres. Three natural polymers were used as packing materials to obtain nanoscale delivery device for BMP, in the presence of phosphatidylcholine functioning as stabilizer. Positively charged polysaccharide, N,N-diethylaminoethyl dextran (DEAE-dextran) tended to form stable, uniform and smaller size particles carrying BMP. Negatively charged bovine serum albumin (BSA) induced precipitation of the produced BMP particles due to its weak interaction with BMP molecules, although it produced nanosized BMP spheres. While collagen, a weakly positively charged protein shaped larger particles due to the strong interaction among themselves. A mechanism of co-precipitation process was also deduced to depict the formation of stable nanospheres.

Towards more efficient glutathione peroxidase mimics: substrate recognition and catalytic group assembly.

Glutathione peroxidase (GPX) is a well-known selenoenzyme that functions as an antioxidant and catalyzes the reduction of harmful peroxide by glutathione and protects cells against oxidative damage. Because many diseases are related to oxidative stress, GPX is an ancient foe of many diseases. Antioxidants are very useful for biological bodies, and considerable effort has been spent to find compounds that could imitate the properties of GPX. This paper reviews GPX mimics developed so far and describes a new, more effective strategy for fabricating them. Although many GPX mimics have been made, they possess serious disadvantages: low activity, low solubility in water, and, in some cases, toxicity. In order to overcome these drawbacks, we have proposed a new strategy of imitating GPX. First, a receptor with a substrate binding site is generated. Next, a catalytic group is incorporated into the receptor near the substrate binding site, allowing the catalytic group access to the functional group of the substrate. Finally, a highly efficient enzyme mimic is obtained. Using this strategy, we successfully fabricated GPX mimics that use antibodies, cyclodextrins, some enzymes and proteins as receptors and chemical modification to incorporate the catalytic group, selenocysteine (Sec). The general principle of combining a functional group involved in catalysis with a specific binding site for the substrate is an approach that could be applied to the generation of other efficient semisynthetic biocatalysts. We describe the antioxidant activities of these GPX mimics and the reasons of their being promising candidates for medicinal applications.