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Amiodarone (AM) is one of the most effective antiarrhythmic drugs and normally administrated by intravenous infusion which is liable to cause serious phlebitis. The therapeutic drugs for preventing this complication are limited. Intermedin (IMD), a member of calcitonin family, has a broad spectrum of biological effects including anti-inflammatory effects, antioxidant activities, and antiapoptosis. But now, the protective effects of IMD against amiodarone-induced phlebitis and the underlying molecular mechanism are not well understood. In this study, the aim was to investigate the protective efficiency and potential mechanisms of IMD in amiodarone-induced phlebitis. The results of this study revealed that treatment with IMD obviously attenuated apoptosis and exfoliation of vascular endothelial cells and infiltration of inflammatory cells in the rabbit model of phlebitis induced by intravenous infusion of amiodarone compared with control. Further tests in vitro demonstrated that IMD lessened amiodarone-induced endothelial cell apoptosis, improved amiodarone-induced oxidative stress injury, reduced inflammatory reaction, and activated the Wnt/β-catenin signal pathway which was inhibited by amiodarone. And these effects could be reversed by Wnt/β-catenin inhibitor IWR-1-endo, and si-RNA knocked down the gene of Wnt pathway. These results suggested that IMD exerted the protective effects against amiodarone-induced endothelial injury via activating the Wnt/β-catenin pathway. Thus, IMD could be used as a potential agent for the treatment of phlebitis.

Various genetically engineered microorganisms have been developed for the removal of heavy metal contaminants. Metal biosorption by whole-cell biosorbents can be enhanced by overproduction of metal-binding proteins/peptides in the cytoplasm or on the cell surface. However, few studies have compared the biosorption capacity of whole cells expressing intracellular or surface-displayed metal-adsorbing proteins. In this study, several constructs were prepared for expressing intracellular and surface-displayed Ochrobactrum tritici 5bvl1 ChrB in Escherichia coli BL21(DE3) cells. E. coli cells expressing surface-displayed ChrB removed more Cr(VI) from aqueous solutions than cells with cytoplasmic ChrB under the same conditions. However, intracellular ChrB was less susceptible to variation in extracellular conditions (pH and ionic strength), and more effectively removed Cr(VI) from industrial wastewater than the surface-displayed ChrB at low pH (

RNA splicing is a fundamental regulatory process of gene expression and plays a pivotal role in transcriptome complexity, cell-fate determination, and organism development. The detection of splicing products has attracted significant interest because aberrant splicing can lead to cell dysfunction and numerous diseases, such as cancer and neurodegeneration. Particularly, splicing variability between individual cells is primarily responsible for gene expression heterogeneity. Investigations into single-cell expression of RNA splicing variants offer a venue for resolving gene regulatory circuits, mapping intracellular localization, and classifying cell types. This review discusses the merits and technical challenges in transitioning RNA splicing detection from in vitro testing to single-cell analysis. In the end, we suggest some future directions for RNA splicing analysis and expect to inspire innovative works and discoveries in this field.

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.

Summary The expression and spatial profile of multiple RNA species at high precision in single cells is key information for understanding cellular behaviors and functions. Fluorescence microscopy is a powerful tool for imaging RNAs, but its multiplexing ability is limited by the number of spectrally distinct fluorophores. Here, we present a DNA-sequence-encoded fluorescence barcoding method, termed sequence-encoded amplicon (SeqEA), enabling highly multiplexed imaging of RNAs in single cells with single-molecule and single-nucleotide resolution. SeqEA exploits a thermodynamically tuning DNA hybridization approach for precisely encoding rolling circle amplicons with discrete fluorescence intensities as fluorescence barcodes, allowing for multiplexed tagging single-molecule RNAs. As a proof of principle, we have demonstrated its ability for simultaneously visualizing nine mRNA species and evaluated the coordination and spatial pattern of breast-cancer-associated oncogene expression. SeqEA provides a single-cell analysis platform for investigating the gene expression network and molecular mechanism of disease development.

While zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases in Escherichia coli Here, we report that the intracellular zinc overload in E. coli cells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene cluster iscSUA-hscBA-fdx-iscX in E. coli cells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of the E. coli mutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin in E. coli cells.IMPORTANCE Zinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload in Escherichia coli cells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

ContextEvidence indicates that there is substantial impairment/loss of β-cell function/mass even before prediabetes. Elevated plasma proinsulin is a sign of β-cell dysfunction in patients with diabetes/prediabetes. However, the dynamic changes of glucose stimulated proinsulin secretion (GSPS) among nondiabetic individuals remain obscure.ObjectiveTo examine GSPS and glucose-stimulated insulin secretion (GSIS) among individuals with normal glucose tolerance (NGT) and impaired glucose tolerance (IGT) and to evaluate whether impaired GSPS is an early biomarker of β-cell impairment in individuals with NGT who have subthreshold postprandial plasma glucose (PPG).Design and ParticipantsWe evaluated GSPS and GSIS in 116 Chinese adults without diabetes (mean age ± SD, 33.31 ± 9.10 years; mean BMI, 25.24 ± 4.20 kg/m2) with fasting plasma glucose (FPG) < 5.6 mmol/L. Based on 2hPPG, the participants were divided into three groups: NGT1 (2hPPG < 6.67 mmol/L), NGT2 (6.67 ≤ 2hPPG < 7.78 mmol/L), and IGT (7.78 ≤ 2hPPGResultsAlthough not diagnosed with prediabetes, the individuals with NGT2 have clinical characteristics and high diabetes risk factors similar to those of the IGT group. However, unlike individuals with IGT, NGT2 participants did not exhibit a delayed GSIS. Instead, GSPS was impaired in NGT2 groups but not in NGT1 group.ConclusionsThis study suggests that impaired GSPS, but not impaired GSIS, may serve as an early biomarker to identify a subpopulation of NGT with a high risk of diabetes.

The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging-associated diseases. Imaging of single-nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9-mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single-molecule resolution. Using CasPLA, we observed a mtDNA-transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy genomic loci ( KRAS gene) in the nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.

This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside and they were purified by Glutathione Sepharose 4B. This protocol can be adapted for the purification of other proteins.

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy. Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages, which results in more peptides that are compatible with mass-spectrometry analysis. Compared to conventional solution digestion, immobilized protease digestion has the obvious advantages of short digestion time, no self-proteolysis, and reusability. We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above. Graphene-oxide (GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface. The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis. Compared to 12-hour solution digestion using trypsin or Glu-C, 14% and 7% improvement were obtained, respectively, in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C. Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3% and 48.7% enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone. The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO, which leads to fewer missed cleavages and more complete digestion. As a result, improved protein identification and sequence coverage can be expected.

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins, and then chelation of lanthanide metal ions as tags, we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry. The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level. The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads. We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75 °C, among which N-terminal peptides of 24 proteins are partially acetylated. Moreover, metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified; these make up 55 percent of the identified proteins. Finally, we developed a general approach for the relative quantification of proteins based on N-terminal peptides. We adopted lysozyme and ribonuclease B as model proteins; the correlation coefficients (R 2) of the standard curves for the quantitative method were 0.9994 and 0.9997, respectively, with each concentration ratio ranging from 0.1 to 10 and both relative standard derivations (RSD) measured at less than 5%. In T. tengcongensis at two incubation temperatures, 80 proteins possess quantitative information. In addition, compared with the proteins of T. tengcongensis incubated at 55 °C, in T. tengcongensis incubated at 75 °C, 7 proteins upregulate whereas 16 proteins downregulate, and most differential proteins are related to protein synthesis.

Protein glycosylation is involved in the control of many important biological processes and structural alterations of the N-linked glycans are correlated with various kinds of disease. High-throughput N-glycan profiling is a key technique for elucidating the functions of glycans in biological process and disease development as well as discovering new diagnostic biomarkers. However, the low abundance of glycans existing in living organism, the competition/suppression effect of other highly abundant biological molecules and the inherent lack of alkalinity and hydrophobicity of glycans leads to particularly poor detection sensitivity in MS analysis. Here, we demonstrated the first “one-step” approach for highly efficient glycan enrichment and derivatization using reduced graphene oxide as nanoreactors and 1-pyrenebutyric hydrazide for glycan capture and derivatization, which resulted in a 33-fold increase in the glycan detection sensitivity in MALDI-TOF-MS and the identification of 48N-glycoforms from human plasma.

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

Telomerase is a nonclassical DNA polymerase that uses its integral RNA as a template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase is unique and involves a translocation step after the synthesis of each telomeric repeat. To directly measure the enzymatic turnover of substrate and the efficiency of the translocation step we have extended our two-color single molecule fluorescence coincidence method (Anal.Chem. 2003, 75, 1664-1670). The method employs Cy5-dATP incorporation into a DNA primer that has been prelabeled with a reference fluorophore. Measurements are performed in the single molecule regime and products, which necessarily have both fluorophores, are excited by two independent lasers, and give rise to coincident events. By counting the number of coincident events and using the coincidence detection efficiency, it is possible to determine the number of the extended products generated by attomole quantities of telomerase, without separation or the use of PCR or radioactivity. Histograms of the logarithms of the ratios of the Cy5 to the reference fluorophore fluorescence can be used to determine the length distribution of the products and hence the enzyme processivity. The mean processivity obtained from the single molecule fluorescence coincidence assay is 0.32 +/- 0.04, in good agreement with the value of 0.37 +/- 0.05 derived from the direct radioactive assay approach. The function of the alignment domain of human telomerase RNA in sustaining catalytic activity in vitro has been reevaluated using this method. Together with our previous results (Nucleic Acids Res. 2002, 30, 4470-4480) these experiments identify the essential residues in the alignment domain of human telomerase RNA that contribute to the activity and processivity of telomerase.

A strategy for design of bioimprinted proteins with glutathione peroxidase (GPX) activity has been proposed. The proteins imprinted with a glutathione derivative were converted into selenium-containing proteins by chemical modifying the reactive hydroxyl groups of serines followed by sodium hydrogen selenide displacement. These selenium-containing proteins exhibited remarkable GPX activities and the GPX activities of reduction of H 2 O 2 by glutathione (GSH) were found to be 101-817 U μmol -1 , which approaches the activity of a selenium-containing catalytic antibody elicited by a hapten similar to our template. The steady state kinetic study for imprinted protein catalysis revealed Michaelis-Menten kinetics for both H 2 O 2 and GSH, e.g. the pesudo-first-order rate constant k cat (H 2 O 2 ) and the apparent Michaelis constant K m (H 2 O 2 ) at 1 mM GSH were calculated to be 784 min -1 and 1.24 × 10 -3 M, respectively, and the apparent second-order rate constant k cat (H 2 O 2 )/K m (H 2 O 2 ) was determined to be 6.33 x 10 5 (M min) -1 . The kinetics and the template inhibition showed that the strategy might be a remarkably efficient one for generating artificial enzyme with GPX activity.

Reactive oxygen species (ROS) are products of normal metabolic activities and are thought to be the cause of many diseases. A selenium-containing single-chain abzyme 2F3 (Se-2F3-scFv) that imitates glutathione peroxidase has been produced which has the capacity to remove ROS. To evaluate the antioxidant ability of Se-2F3-scFv, we constructed a ferrous sulfate/ascorbate (Vc/Fe2+)-induced mitochondrial damage model system and investigated the capacity of Se-2F3-scFv to protect mitochondria from oxidative damage. Se-2F3-scFv markedly decreased mitochondrial swelling, inhibited lipid peroxidation, and maintained the activity of cytochrome c oxidase, in comparison with Ebselen, a well-studied glutathione peroxidase mimic, indicating that Se-2F3-scFv has potential for treating diseases mediated by ROS.

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.

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 microM(-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.

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.

A novel strategy for design of haptens that were used to produce catalytic antibodies was developed and three monoclonal antibodies, 3G5, 2F3, and 5C9, were generated using this strategy. These monoclonal antibodies were converted into selenium-containing abzymes by chemically modifying the hydroxyl group of serines followed by sodium hydrogen selenide displacement. These selenium-containing abzymes exhibited remarkable glutathione peroxidase activity, which surpasses the activity of some native glutathione peroxidases. The activities of the selenium-containing abzymes Se-3G5, Se-2F3, and Se-5C9 which catalyzed reduction of hydroperoxides by glutathione were 2.23, 4.20, and 3.79 times that of rabbit liver glutathione peroxidase, respectively. Detailed steady-state kinetics study on Se-2F3 was carried out and the value of k(cat)/K(m) (H(2)O(2)) was found to be 2.11 x 10(7) M(-1) min(-1) which was supposed to be one of the highest among the known catalytic antibodies. The data of association constants and glutathione peroxidase activities of these catalytic antibodies and the steady-state kinetics of Se-2F3 showed that the method might be a remarkably efficient one for generating catalytic antibodies with glutathione peroxidase activity.

The steady-state kinetics study and some enzymatic characterization of a selenium-containing scFv catalytic antibody (Se-scFv2F3) were carried out. A novel reaction formula of this abzyme-catalyzed reaction was proposed and a rate equation was obtained according to the formula. The constants in the equation were compared with Dalziel's parameters and the exact meanings of these constants were analyzed. The obtained kinetics parameters from the kinetics study of Se-scFv2F3 were analyzed and compared with those of native glutathione peroxidase.

A diselenide, 2,2'-diseleno-bis-beta-cyclodextrin (2-SeCD), was synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). The GPX mimic accepts a variety of hydroperoxides as substrates. The GPX activities, reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, are 7.4, 4.5 and 10.2 U/micromol, respectively. In contrast to ebselen (PZ51), the diselenide displays high GPX-like activity. The reduction of hydroperoxide by glutathione in the presence of a radical trap shows that the mimic catalyzes the reaction via a non-radical mechanism. A ping-pong mechanism was observed in the steady-state kinetic studies of the 2-SeCD-catalyzed reaction.

A novel artificial glutathione peroxidase mimic consisting of a selenocystine-di-beta-cyclodextrin conjugate (selenium-bridged-6, 6'-amino-selenocystine-6,6'-deoxy-di-beta-cyclodextrin), in which selenocystine is bound to the primary side of beta-cyclodextrin through the two amino nitrogen groups of selenocystine, was synthesized. The glutathione peroxidase activities of the mimic-catalyzed reduction of H(2)O(2), tert-butylhydroperoxide, and cumene hydroperoxide by glutathione are 4.1, 2.11, and 5.82 units/micromol, respectively. The first activity was 82 and 4.2 times as much as that of selenocysteine and ebselen, respectively. Studies on the effect of substrate binding on the glutathione peroxidase activity suggest that it is important to consider substrate binding in designing glutathione peroxidase mimics. The detailed steady-state kinetic studies showed that the mimic-catalyzed reduction of H(2)O(2) by glutathione followed a ping-pong mechanism, which was similar to that of the native glutathione peroxidase.

Human telomerase is a ribonucleoprotein that is minimally comprised of protein (hTERT) and RNA (hTR) components. We have applied single-molecule fluorescence two-color coincidence detection to characterize complex formation between fluorophore-labeled components in solution. By systematic labeling and in vitro assembly of hTERT, hTR and telomerase's DNA substrate, we have established that catalytically functional human telomerase comprises a stable hTERT:hTR:substrate interaction in a 1:1:1 absolute stoichiometry.

Single-molecule fluorescence has the capability to detect properties buried in ensemble measurements and, hence, provides new insights about biological processes. Ratiometric methods are normally used to reduce the effects of excitation beam inhomogeneity. Fluorescence resonance energy transfer is widely used but there are problems in inserting the fluorophores in the correct position on the biomolecule, particularly if the structure is not known. We have recently developed two-colour coincidence single-molecule fluorescence that addresses this problem. This method can be used to determine quantitatively the multimerization states of biomolecules, in solution without separation. The future prospects of single-molecule fluorescence as applied to biological molecules are discussed.

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.

Cell proliferation and cell death of keratinocytes are tightly regulated to ensure epidermal homeostasis. UV-B induces keratinocyte apoptosis. UV-B also induces lipid peroxidation of keratinocytes to increase their amount of malondialdehyde (MDA). These phenomena can be explained by the production of reactive oxygen species (ROS) induced by UV-B radiation. We synthesized 2-selenium-bridged β-cyclodextrin (2-SeCD) to imitate glutathione peroxidase (GPX), an important antioxidant and established a damage system, in which keratinocytes can be damaged by Ultraviolet B (UV-B) radiation. Using this damage system we studied 2-SeCD protection of keratinocytes against injury induced by UV-B. Experimental results showed that 2-SeCD could protect keratinocytes from apoptosis. Moreover, 2-SeCD inhibits lipid peroxidation of keratinocytes and scavenges ROS. 2-SeCD inhibits the UV-B induced apoptotic signal transduction. This antiapoptotic mechanism may be partly related to the elimination of hydrogen peroxide.

Protein N-glycosylation plays an important role in a variety of biological processes, and unusual changes in glycan structures are often correlated with various pathological states. Therefore, N-glycan profiling not only plays a vital role in the mechanism study of biological processes but also contributes to the discovery of biomarkers for early disease diagnosis. However, the conventional in-solution release of N-glycans by free endoglycosidases, such as peptide N-glycosidase F (PNGase F), suffers from several drawbacks, including prolonged incubation time, possible incomplete digestion and high cost due to the non-reusability of the enzyme. Herein, we report a novel graphene oxide (GO)-based immobilized PNGase F reagent for fast and highly efficient N-glycan release and glycoform profiling of glycoproteins. To create this reagent, PNGase F was attached to the GO surface via carbodiimide-activated amidation. The ultra-high surface area of GO allows for high loading with PNGase F and significantly facilitates the deglycosylation process. Highly efficient N-glycan release of ribonuclease B and asialofetuin was achieved with this reagent within 2 min. Furthermore, the successful application of our GO-based immobilized PNGase F in the N-glycan release of the human plasma glycoproteome was demonstrated by reproducible N-glycan identification using MALDI-TOF MS.

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 antibody's 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.