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Multimetallic nanoframes (NFs) with large specific area and unsaturated coordination sites showed superior catalytic performance in electrochemical catalysis. Herein, we synthesized robust trimetallic PtRuCu NFs with high shape selectivity via preferential etching of Cu in PtRuCu nanoparticles (NPs) with nitrosonium tetrafluoroborate (BF4NO). As far as we know, this is the first time that BF4NO has been used as an etching reagent. PtRuCu NF/C exhibited superior mass activity and specific area activity over commercial Pt/C towards the methanol oxidation reaction (MOR) due to the large specific area, high-density low-coordinated sites of the frame nanostructures and the synergistic effect of the trimetallics. Moreover, PtRuCu NFs/C also showed higher catalytic activity than the similar NFs obtained with acetic acid (HAc) as the etching reagent. We attributed the better performance to the fact that BF4NO was not only an etching-agent but also a ligand exchange reagent that could remove the olamine ligands on the catalyst surface, thus releasing more catalytic sites to improve the catalytic performance.

For the first time, shape-tunable Pt-Ir alloy nanocatalysts including both single-crystalline (nano-octahedra (NOs), nano-truncated octahedra (NTOs), nanocubes (NCs)) and polycrystalline (nanocluster flowers (NCFs), nanowires (NWs), nano-short-chains (NSCs), and nano-octahedral stars (NOSs)) ones were synthesized with a facile one-pot solvothermal method, via precise control of the facet-selective agents (Br- and I-). The surface effects of Pt-Ir alloy nanocatalysts for oxygen electrode reaction in acidic solution were intensively investigated. Pt-Ir alloy nanocatalysts showed enhanced catalytic activities for the oxygen evolution reaction (OER), which were 1.6 to 2.0 times those of the commercial Ir/C catalyst and the Pt/C-Ir/C mixture at an overpotential of 0.25 V. The catalytic activity for the OER exhibited a positive correlation with the proportion of surface IrOx species, but was restricted by the surface alloying effect. Besides the change of the intermediate adsorption state, the dissociation of water was also confirmed to be effective as the rate-determining step of the Pt-Ir alloy nanocatalysts. The catalytic activity for the oxygen reduction reaction (ORR) decreased with the increase of surface IrOx species. Pt-Ir nano-short-chains (NSCs) exhibited 1.3 times the catalytic activity as that of the commercial Pt/C catalyst at 0.80 V and 0.85 V, owing to the higher proportion of the (110) facets with irregular step sites exposed after the annealing treatment at 350 °C. The unique structure could prevent the mass transfer process from being obstructed by adsorbed bisulfate anions and oxidized species on the surfaces. Pt-Ir NSCs exhibited a catalytic efficiency of 46.7% and were considered to be a promising URFC catalyst.

We report a facile one-pot solvothermal synthesis of monodisperse iridium (Ir) ultrasmall (1.5–2.5 nm in diameter) nanoparticles (NPs), worm-like chain nanowires (NWs), and porous nanodendrites (NDs), for which CO oxidation reaction has been employed as a probe reaction to investigate the effects of nanoparticle size and surface-capping organics on the catalytic activities. Time-dependent experiments revealed that an oriented attachment mechanism induced by the strong adsorption of halide anions (Br− and I−) on specific facet of Ir nanoclusters or by decreasing the reduction rate of Ir precursors with changing their concentrations during the synthesis was responsible for the formation of Ir NWs and NDs. Annealing tests indicated that an O2-H2 atmosphere treatment turned out to be an effective measure to clean up the surface-capping organics of Ir NPs supported on commercial SiO2. Catalytic CO oxidation reaction illustrated that a significant improvement in the catalytic activity of CO oxidation reaction was achieved together with the changing of activation energies after such atmosphere treatment for the supported catalysts of the ultrasmall Ir NPs. It is noteworthy that this enhancement in catalytic activity could be ascribed to the changes in the surface status (including populations of Ir species in metallic and oxidized states, removal of surface capping organics, the variety of active sites, and total effective active site number) for the supported nanocatalysts during the atmosphere treatment.

The fabrication of atom-layered two dimensional (2D) noble metal nanosheets (NSs) in a face-centered cubic (fcc) structure is of broad scientific and technological importance, yet this remains a challenge due to the intrinsic cubic symmetry and high surface energy of fcc noble metals. Herein, we report a solid-liquid interface mediated 2D growth method towards the synthesis of hierarchically-coiled ultrathin Ir NSs (thickness

The magnetic gelatin-starch microspheres were prepared by modified emulsion cross-linking method with glutaraldehyde as the cross-linking agent. The structure, size distribution as well as morphology of magnetic microspheres were investigated by FT-IR spectrometer, dynamic laser scattering analyzer and scanning electron microscope, respectively. Bovine serum album (BSA) was chosen as model protein, and the adsorption processes were carried out under diversified conditions including BSA initial concentration, pH value, adsorption time and temperature to evaluate the performance of the magnetic microspheres. The average diameter of optimized spherical magnetic microspheres is 1.6 μm with excellent dispersivity, and the saturation magnetization is found to be equal to 1.056×10−2 A·m2. The adsorption isotherm of the BSA on the magnetic microspheres basically obeys the Langmuir model, with a maximum adsorption capacity of 120 mg/g and an adsorption equilibrium constant of 1.60 mL/mg.

We demonstrate a general strategy for the highly selective synthesis of Rh-based multi-metallic nanoframes through preferential etching of 3d metals, including Cu and Ni. Compared with Rh-Cu nanooctahedrons/C, Rh-Cu nanooctahedral frames/C show greatly enhanced activity toward hydrazine decomposition at room temperature.

5-Aminolevulinic acid (5-ALA) has been suggested for improving plant salt tolerance via exogenous application. In this study, we used a transgenic canola (Brassica napus), which contained a constituted gene YHem1 and biosynthesized more 5-ALA, to study salt stress responses. In a long-term pot experiment, the transgenic plants produced higher yield under 200 mmol L(-1) NaCl treatment than the wild type (WT). In a short-term experiment, the YHem1 transformation accelerated endogenous 5-ALA metabolism, leading to more chlorophyll accumulation, higher diurnal photosynthetic rates and upregulated expression of the gene encoding Rubisco small subunit. Furthermore, the activities of antioxidant enzymes, including superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase, were significantly higher in the transgenic plants than the WT, while the levels of O2 ·(-) and malondialdehyde were lower than the latter. Additionally, the Na(+) content was higher in the transgenic leaves than that in the WT under salinity, but K(+) and Cl(-) were significantly lower. The levels of N, P, Cu, and S in the transgenic plants were also significantly lower than those in the WT, but the Fe content was significantly improved. As the leaf Fe content was decreased by salinity, it was suggested that the stronger salt tolerance of the transgenic plants was related to the higher Fe acquisition. Lastly, YHem1 transformation improved the leaf proline content, but salinity decreased rather than increased it. The content of free amino acids and soluble sugars was similarly decreased as salinity increased, but it was higher in the transgenic plants than that in the WT.

Aqueous dispersion and stability of Fe3O4 nanoparticles remain an issue unresolved since aggregation of naked iron nanoparticles in water. In this study, we successfully synthesized different Fe3O4 super-paramagnetic nanoparticles which were modified by three kinds of materials [DSPE-MPEG2000, TiO2 and poly acrylic acid (PAA)] and further detected their characteristics. Transmission electron microscopy (TEM) clearly showed sizes and morphology of the four kinds of nanoparticles. X-ray diffraction (XRD) proved successfully coating of the three kinds of nanoparticles and their structures were maintained. Vibrating sample magnetometer (VSM) verified that their magnetic properties fitted for the super-paramagnetic function. More importantly, the particle size analysis indicated that Fe3O4@PAA had a better size distribution, biocompatibility, stability and dispersion than the other two kinds of nanoparticles. In addition, using CNE2 cells as a model, we found that all nanoparticles were nontoxic. Taken together, our data suggest that Fe3O4@PAA nanoaparticles are superior in the application of biomedical field among the four kinds of Fe3O4 nanoparticles in the future.

Asymmetric Michael addition of cyclohexanone to nitrostyrenes in the presence of organocatalyst 1 (10 mol-%) and 2,4-dichlorobenzoic acid (10 mol-%) afforded the corresponding synthetically valuable γ-nitroketones in moderate to good yields with high diastereoselectivities (up to >99:1 dr) and high enantioselectivities (up to >99 % ee) under mild conditions.

CobB is a bacterial NAD(+)-dependent protein deacetylase. Although progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear. Using proteome microarray technology, potential substrates of Escherichia coli CobB were screened and nine proteins were identified, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by western blotting and mass spectrometry, and two acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of each acetylated lysine decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at the two acetylated lysine residues are involved in both the O-acetyltransferase and N-acetyltransferase activity of NhoA. Structural analyses were also performed to explore the effects of the acetylated lysine residues on the activity of NhoA. These results suggest that reversible acetylation may play a role in the activity of Escherichia coli NhoA.

Based mainly on SiC aggregate and fine powder, silicon powder, flake graphite (300 μm, 45 μm), α-Al2O3 micro-powder and AlN powder, Graphite-SiAlON bonded SiC composite were prepared. The influence of graphite addition, which varied from 0 to 9.7%, and its grain size on the oxidation resistance was investigated at 1 150 °C under air atmosphere. The results show that: 1) mass change rate reduces with the increase of graphite addition, the mass change rate of sample with 45 μm graphite is higher than that of sample with 300 μm graphite; 2) the cold modulus of rupture of sample with 45 μm graphite after oxidation is higher than that of sample with 300 μm graphite; 3)the oxidation resistance declines along with the increase of graphite addition, additionally, the oxidation resistance of sample with 45 μm graphite is superior to that of sample with 300 μm graphite; 4) 7.5% graphite is recommended as the proper addition.

Based on aluminum powder, Silicon powder, activated alumina and flake graphite as the main starting materials, SiAlON-Graphite composites were produced in N2 atmosphere. The influence of firing temperature and atmosphere on Si-Al-O-C-N system was investigated. It is proposed that the introduction of graphite produces a certain CO partial pressure and affects the nitridation of metal silicon, therefore reducing the mass change. In addition, large amount of 15R are formed through carbothermic reduction. CO hinders the growth of β-SiAlON. Z value of β-SiAlON in sample C with graphite is less than sample S without graphite.

A gene encoding 5-aminolevulinic acid synthase (ALA-S) in Saccharomyces cerevisiae (Hem1) under the control of an Arabidopsis thaliana HemA1 promoter (HemA1 promoter) was introduced into the genome of tomato (Solanum lycopersicum). All transgenic tomato plants were able to transcribe the Hem1 gene and the capacity to synthesize ALA was increased. The transgenic plants had 18% higher levels of chlorophylls, as compared to wild-type plants, indicating that part of the additional ALA from yeast ALA-S was metabolized to chlorophylls. Determination of gas exchange suggested that the net photosynthetic rate (Pn) in the transgenic tomato was about 60% higher than that of wild type. The apparent quantum yield (AQY) in the transgenic plants was 0.0363, significantly higher than 0.0313 of wild type. The Chlorophyll-a fast fluorescence measurement and JIP-test at continuous intensities of excitation light (90-2100 μmol m-2 s-1) showed that the genetic transformation caused a significant reduction in Vj (the relative variable fluorescence at the J-step) but addition in Vi (the relative variable fluorescence at the I-step). The maximum photochemical efficiency (φPo=Fv/Fm) did not vary with the light intensity, which demonstrated that φPo was a stable index for leaf photosynthesis. ψo (the probability that a trapped exciton moves an electron into the electron transport chain beyond QA-) and φEo (the quantum yield of electron transport) decreased with the light intensity increased, especially in wild type. Furthermore, the photosynthetic performance index on absorption basis (PIABS) or cross section basis (PICS) in the transgenic plants was also increased obviously in transgenic tobacco. Thus, the results obtained in this work approved that ALA improved photosynthetic capacity and the activity of PSII reaction center.

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

Strain JAU4234, identified as Streptomyces padanus , was isolated from soil collected in Jiangxi Province, China. It produced actinomycin X2, fungichromin, and a new polyene macrolide compound with antifungal activity, antifungalmycin 702. Antifungalmycin 702 had good general antifungal activity and may have potential future agricultural and/or clinical applications.

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)β(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.

Application with 50 mg L−1 5-aminolevulinic acid (5-ALA) to pakchoi (Brassica campestris ssp. chinensis var. communis Tsen et Lee) seedlings showed that when seedlings were cultured in 1/2 Hoagland's solution containing 1/2 of standard (3.75 mmol L−1) or standard nitrate (7.5 mmol L−1), the outer leaves of rosette plants gradually turned yellow, whereas much fewer yellowed leaves were found when 5-ALA was added. Analysis showed that 5-ALA treatment increased the leaf chlorophyll content, root activities, and insomuch plant production. 5-ALA treatment also greatly increased the content of free amino acids and soluble proteins. However, 5-ALA treatment decreased nitrate accumulation in both leaf blades and petioles, which was related with higher activity of nitrate reductase (NR) and higher level of NR gene transcript in pakchoi leaves. These results suggest that exogenous 5-ALA treatment may promote nitrogen absorption and assimilation with less nitrate accumulation in leaf tissues.

Although matrine, a primary active component of dried Sophora flavescens root (ku shen), is known to induce apoptosis in a variety of tumor cells in vitro, the molecular mechanism of such apoptosis remains elusive. This analysis of the cell cycle and apoptosis in matrine-treated human gallbladder carcinoma cells (GBC-SD) showed that matrine can indeed inhibit cell proliferation and induce G1 cell cycle arrest and apoptosis in a dose- and time-dependent manner. An additional western blot analysis of matrine-treated cells also showed caspase-3 and Bcl-2 activation, as well as cyclinE down-regulation. Overall, the results indicate that matrine perturbs gallbladder cancer cell progression during the G1 phase by down-regulating cyclinE and induces apoptosis by decreasing the expression of the antiapoptotic protein Bcl-2 and increasing expression of the proapoptotic protein Bax.

Surface monofunctionalization of protein nanostructures will enable precise topological control over the protein-templated assembly of nanoscale motifs, however, this remains a formidable challenge. Here we demonstrated a novel strategy for this purpose with a protein nanocage, virus-based nanoparticle (VNP) of simian virus 40 as a model system. By simultaneously incorporating a function modality (cysteine) and a purification modality (polyhistidine tag) into the building block (VP1) of VNPs through rational design and genetic engineering, the monofunctionalized cysteine-VNPs are readily obtained through a routine affinity chromatography in virtue of the purification modality of polyhistidine tag, after the coassembly of the functional VP1 and the nonfunctional VP1 at an optimal ratio. This strategy has proved to be highly efficient in constructing monofunctionalized protein nanostructures as highlighted by the monofunctionalized-VNP-guided Au/QD-VNP nanostructures. These nanostructures could be utilized in a wide range of disciplines, including basic biological research, novel nanostructures, and nanodevices fabrication, etc.

Based on SiC grains and powder, flake graphite, AlN powder, Silicon powder, sintered alumina ultra-fine powder as the starting materials, the sample of SiAlON-Graphite-SiC composites was prepared by firing under N2 atmosphere at 1 550°C and then analyzed in terms of high temperature performances by XRD, SEM and EDAX etc. The interrelation between composition, structure and performance of the material was also investigated. It indicates that this material provides excellent thermal shock resistance and molten alkali resistance, also proper oxidation resistance and applicable as the inner lining of the blast furnace.

Green fluorescent protein (GFP) plasmid was caged by 8-bromo-7-hydroxyquinolinyl chromophore (BHQ) for controlling its expression with exact spatiotemporal resolution. In vitro and in vivo experiments clearly verified that, comparing with Bhc caging, the expression level of caged GFP plasmid was dramatically decreased and then efficiently restored after subsequent photolysis.

In the search for a new Cys side-chain protecting group that is compatible to the solid-phase peptide synthesis yet can be removed under mild conditions, the Hqm and Hgm groups that are readily deprotected by using aqueous hydrazine have been developed. The utility of these groups for peptide and protein chemistry is tested by the total synthesis of a peptide antibiotic trifolitoxin and the human neutrophil defensin hNP2.

A sensitive assay for melamine in complex matrices is built using cysteamine-modified gold nanoparticles (AuNPs) and an effective sample pretreatment protocol. Citrate-stabilized AuNPs were modified by cysteamine in order to weaken the electrostatic repulsion force between the gold nanoparticles. Detection sensitivity gained through this modification increased about 100 fold compared with the result using the unmodified AuNPs. Direct colorimetric visualizations of melamine in milk products, eggs and feeds was successfully demonstrated within the linear ranges of 1-200 mg L(-1) and detection limits below 1 mg L(-1). The proposed scheme could be an alternative means for onsite detection of melamine without costly instruments.

In order to obtain an ultra-sensitive molecular biosensor, we designed an auto-biotinylated bifunctional protein nanowire (bFPNw) based on the self-assembly of a yeast amyloid protein, Sup35, to which protein G and a biotin acceptor peptide (BAP) were genetically fused. These auto-biotinylated bFPNws can transfer hundreds of commercially available diagnostic enzymes to an antigen-antibody complex via the biotin-avidin system, greatly enhancing the sensitivity of immune-biosensing. Compared to our previously reported seeding-induced bFPNws (Men et al., 2009), these auto-biotinylated bFPNws gave greater signal amplification, reduced non-specific binding and improved stability. The auto-biotinylated self-assembled bFPNw molecular biosensors were applied to detect Yersinia pestis (Y. pestis) F1 antigen and showed a 2000- to 4000-fold increase in sensitivity compared to traditional immunoassays, demonstrating the potential use of these self-assembling protein nanowires in biosensing.

The rate constants of the nucleophilic reactions between amines and benzhydrylium ions were calculated using first-principles theoretical methods. Solvation models including PCM, CPCM, and COSMORS, as well as different types of atomic radii including UA0, UAKS, UAHF, Bondi, and UFF, and several single-point energy calculation methods (B3LYP, B3P86, B3PW91, BHANDH, PBEPBE, BMK, M06, MP2, and ONIOM method) were examined. By comparing the correlation between experimental rate constants and the calculated values, the ONIOM(CCSD(T)/6-311++G(2df,2p):B3LYP/6-311++G(2df,2p))// B3LYP/6-31G(d)/PCM/UFF) method was found to perform the best. This method was then employed to calculate the rate constants of the reactions between diverse amines and diarylcarbenium ions. The calculated rate constants for 65 reactions of amines with diarylcarbenium ions are in agreement with the experimental values, indicating that it is feasible to predict the rate constant of a reaction between an amine and a diarylcarbenium ion through ab initio calculation.

In this study, the anticancer effect of icariin, a natural flavonol glycoside, against human hepatoma SMMC-7721 cells and the underlying mechanisms were investigated. Icariin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, icariin induced a sustained activation of the phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 and ERK1/2, and SP600125 (an inhibitor of JNK) almost reversed icariin-induced apoptosis in SMMC-7721 cells. In addition, icariin provoked the generation of reactive oxygen species (ROS) in SMMC-7721 cells, while the antioxidant N-acetyl cysteine almost completely blocked icariin-induced JNK activation and apoptosis. Taken together, these findings suggest that icariin induces apoptosis through a ROS/JNK-dependent mitochondrial pathway.

A thermophilic esterase, SsoPEst, from Sulfolobus solfataricus P2 was cloned and expressed in E. coli AD494 (DE3). Gene sequencing indicated the encoded 353 amino acids had less than 32% identity with reported esterases. The recombinant enzyme hydrolyzed p-nitrophenyl esters but not tributyrin or tricaprylin, exhibiting the highest specific activity (1.1 U/mg) with p-nitrophenyl caprylate. The enzyme was optimally active at pH 5.5 and 80 degrees C. It retained 50% activity after 1 h incubation at 80 degrees C. Activity was significantly inhibited by PMSF. Five SsoPEst mutants were generated by site-directed mutagenesis. One mutant had a higher specific activity of 2.8 U/mg at 37 degrees C and 14 U/mg at 80 degrees C than the wild-type enzyme which exhibited 0.7 U/mg at 37 degrees C and 3.8 U/mg at 80 degrees C against p-nitrophenyl butyrate.

The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14–22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.

A functionalized gold nanoparticle (GNP) enhanced ultrasensitive immuno-PCR assay (GNP-IPCR on ELISA plate), which was modified from the recent developed bio-barcode assay (BCA) technique, was developed to detect Hantaan virus nucleocapsid protein (HNP). During the assay, the target antigen HNP was captured by a polyclonal antibody coated on ELISA microplate wells, followed by adding GNP dually modified with oligonucleotides and a HNP specific monoclonal antibody L13 (mAb L13) to form a sandwich immuno-complex. The oligonucleotides on the GNP contained two strands: one as capture DNA immobilized on the surface of the GNP through Au-S bond and the other as signal amplification DNA, which was partially complementary with the capture DNA. After the immuno-complex was formed, the signal DNA was released by heating, and consequently characterized by PCR/gel electrophoresis and SYBR-Green real time PCR. The detection limit of this method could reach down to 10 fg/mL for detecting purified HNP in buffers as well as in human serum, which was approximately 7 orders of magnitude more sensitive than that of conventional ELISA. The current assay format might be adopted for other proteins that need ultra-high sensitive detection.

This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia coli alkaline phosphatase (EAP) is a well-defined nonspecific phosphated monoesterase and Ser-, Thr- or Tyr-phosphorylated peptides served as substrates for EAP in preliminary experiments. Based on the known catalytic mechanism of EAP, the recombinant site-directed mutant EAP-S102L was generated, whose catalytic activity was blocked, but its binding ability was preserved. For EAP-S102L the catalytic rate constant, k(cat), was reduced by a factor of 1000, while the Michaelis-Menten constant, K(m), remained almost unchanged. Crystallographic analysis of the EAP-S102L/phophorylated peptide complex revealed that EAP-S102L could bind the phosphate group of the phosphorylated peptide but lacked nucleophilic attack potential which was essential for the catalytic ability of EAP. Finally, by combining the fluorescence-labeled EAP-S102L with non-phophorylated peptide chips, kinases could be detected from tumor cell samples. The recombinant EAP-S102L construct is perhaps the first functional binding protein derived from a native enzyme, illustrating how one single mutation tremendously alters protein function.

The tegument protein pp28 of human cytomegalovirus (HCMV) is essential for the assembly of infectious HCMV virions, but how it functions during the process of HCMV tegumentation and envelopment remains unclear. By using live cell fluorescence resonance energy transfer (FRET) microscopy and yeast two-hybrid assays, we found that another HCMV tegument protein, UL94, was a specific binding partner for pp28. The interaction between pp28 and UL94 was imaged in a punctuate, juxtanuclear compartment, previously designated as the virus assembly compartment (AC). Amino acids 22–43 of pp28 were identified as being responsible for its binding with UL94, while no linear binding site could be found within UL94. The interaction between pp28 and UL94 may serve as a link in the sequential processes of HCMV capsidation, tegumentation and envelopment. This study provides a foundation for further studies into how the HCMV tegument proteins act in the assembly of HCMV virions.

The activity of Botulinum neurotoxin type A (BoNT A) can be measured by monitoring the toxin's endopeptidase reaction with its peptide substrate. In this report, a sensitive and simple capillary electrophoresis (CE) method for analysing BoNT A activity was developed using a peptide substrate labelled with Fluorescein isothiocynate (FITC) at the N-terminal and biotin at the C-terminal. This dual labelling enables not only highly sensitive laser induced fluorescence (LIF) detection of the reaction product, but also good analytical separation of the product from the peptide substrate by Micellar Electrokinetic Chromatography (MEKC). The separation between the product peak and the substrate peak was approximately 5 min using the dual-labelled substrate, while just about 1 min using the FITC-labelled substrate without biotinylation. Using the current assay method, BoNT A with concentration as low as 0.1 ng ml−1 (3.6 U mL−1 in mouse LD50) in water was detected with a S:N ratio of 3 (RSD

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 microL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. [figure: see text]

Nucleic acid samples with high concentrations of salt could be stacked and well separated during capillary zone electrophoresis (CZE) by adding glycerol into the samples and using a Tris-Borate-EDTA (TBE) buffer (pH 8.3) as the separation medium. The so-called glycerol-salt mediated stacking was found applicable to different types of nucleic acids. Three nucleic acids: a 16s rRNA (1,542 nt), a double stranded DNA (1.6 k bp), and a single stranded DNA (30 nt), were tested as demos in the experiments. When the sample matrix contained 50 mM KCl and 50% (w/v) glycerol, the 16s rRNA sample could be stacked as high as 30 times compared with the sample without KCl. All the nucleic acids could be stacked effectively when high concentrations of glycerol (>50%) and salt (more than 50 mM) were present in the sample matrix, while the dsDNA could be stacked with high concentrations of glycerol (>50%) alone.

The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 degrees C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn2+/Fe2+-containing protein with a molar ratio of [Mn2+] to [Fe2+] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA.

N,O-Bidentate compounds were systematically evaluated as phosphine-free ligands for Pd-catalyzed C-C bond-formation reactions through kinetic measurements. Pd(quinoline-8-carboxylate)2 was identified as one of the most efficient, yet still low-priced, phosphine-free catalysts for Heck as well as Suzuki reactions of unactivated aryl bromides with high turnover numbers up to ca. 10,000.

Fluorescent photoswitching molecules in which photochromic diarylethene with terpyridine units (tpy) attached via a phenylene linker to the thiophene rings were synthesized. Both compounds exhibited pronounced photochromic properties. Irradiation of the open form of compounds 1 and 2 with UV light resulted in immediate photocyclization to the blue closed form; the colorless open forms could be regenerated by irradiation with visible light. In terms of potential use for optical data storage and readout, compounds 1 and 2 display fluorescence discrimination between their open and closed form. This fluorescence switching could be the basis of a nondestructive readout method. And the complexation of these two compounds with metal ions was also studied. It is amazing to find the addition of Zn 2+ lead the emission wavelengths of 2a red shift up to 105 nm. This unique phenomenon makes the compound 2a , a candidate for the chemical sensor for zinc.

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.

A -cyclodextrin dimer tethered by photoswitchable dithienylethene moieties was synthesized as a potentially tunable receptor. The dimer exhibits pronounced photochromic properties. Irradiation of the dimer in open form with UV light at 254 nm resulted in immediate photocyclization to the pink closed form; the colorless open form could be regenerated by irradiation with visible light of wavelength >460 nm. The reaction kinetics of the forward photoprocess were also studied. To investigate the binding ability of the dimer in open and closed forms, a fluorescence titration was performed. It was found that the stability constant for the binding of TSPP (meso-tetrakis (4-sulfonatophenyl)porphyrin) by the closed form of the dimer is a factor of 5 higher than that of the open form.

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.

1,3-Dipolar cycloaddition of DTE-azomethine ylides (DTE: dithienyl-ethene) to C60 in refluxed toluene was used to synthesize novel dumbbell-type fullerene dimer 1. For the sake of comparison, the monoadduct 2 were also synthesized. The molecular geometries of these two compounds were determined by theoretical calculations with HF-3/21G method. UV-Vis and fluorescence experiments were carried out in solvents with different polarity at the room temperature. All the results indicated the existence of a photoinduced intramolecular electron transfer process between the donor and acceptor moieties.

This study aims to develop a rapid and sensitive cell-free bioassay of dioxins. It is known that dioxin ligand can bind heterodimeric aryl hydrocarbon receptor (AhR) and triggers the formation of the complex of dioxin-AhR, AhR nuclear translocator (ARNT), and dioxin-responsive element (DRE) region of the DNA. The hypothesis of the proposed method is that if FITC were labeled at the DRE sequence, its fluorescence intensity would be enhanced when the complex forms because the interaction interface of the binding components (AhR, ARNT, and DRE) creates a rather hydrophobic condition that is in favor of FITC emission. Effects of modification site of FITC on the DNA probes on binding efficiency between the complex components and fluorescence emission enhancement were evaluated by surface plasmon resonance and fluorescence analysis, respectively. Results showed that the labeling site at the second base at the 5' end apart from the core region (5'-TNGCGTG-3') of DRE did not obviously interfere with the binding between the DNA probe and dioxin-AhR/ARNT hybrid but presented significant fluorescence emission enhancement. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the typical toxin in this study. The method had a linear range of 1-100 pM, with detection limit of 0.1 pM (0.64 fg/assay) and coefficient of variation of 5.6% (n = 10, 50 pM TCDD in transformed cytosol). The whole detection cycle was approximately 4 h. The method was also used to estimate the toxic equivalents (TEQ) of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD). Measurement of TEQs of the mixture of TCDD, PeCDD, and HxCDD were highly consistent with the predicted data. The average recovery using fly ash extract was approximately 93%.

A number of naphthalene derivatives containing adamantanamine binding moiety and an (CH2)n (n=2, 3, 4, 5, 6) spacer were prepared as the electron donor. A supramolecular assembly was fabricated by the inclusion between the donor substrates and the host molecules, i.e., mono-6-O-p-nitrobenzoyl-β-cyclodextrin (pNBCD) and mono-6-O-m-nitrobenzoyl-β-cyclodextrin (mNBCD), in water. The fluorescence quenching in these systems was studied in detail. It revealed efficient photoinduced electron transfers (PET) between the naphthalene donors and the cyclodextrin acceptors. This PET process was partitioned into a dynamic quenching component caused by bimolecule collision reactions and a static quenching component due to hydrophobic binding between the donor and acceptor molecules. Detailed Stern–Volmer constants were measured and they were partitioned into dynamic Stern–Volmer quenching constants (dynamic quenching) and static binding constants (static quenching). In these two pathways, the static quenching was found to be highly efficient and dominant in the presence of NBCD.

Methyl parathion hydrolase (MPH, E.C.3.1.8.1), isolated from the soil-dwelling bacterium Pseudomonas sp. WBC-3, is a Zn(II)-containing enzyme that catalyzes the degradation of the organophosphate pesticide methyl parathion. We have determined the structure of MPH from Pseudomonas sp. WBC-3 to 2.4 angstroms resolution. The enzyme is dimeric and each subunit contains a mixed hybrid binuclear zinc center, in which one of the zinc ions is replaced by cadmium. In both subunits, the more solvent-exposed beta-metal ion is substituted for Cd2+ due to high cadmium concentration in the crystallization condition. Both ions are surrounded by ligands in an octahedral arrangement. The ions are separated by 3.5 angstroms and are coordinated by the amino acid residues His147, His149, Asp151, His152, His234 and His302 and a water molecule. Asp255 and a water molecule serve to bridge the zinc ions together. MPH is homologous with other metallo-beta-lactamases but does not show any similarity to phosphotriesterase that can also catalyze the degradation of methyl parathion with lower rate, despite the lack of sequence homology. Trp179, Phe196 and Phe119 form an aromatic cluster at the entrance of the catalytic center. Replacement of these three amino acids by alanine resulted in a significant increase of K(m) and loss of catalytic activity, indicating that the aromatic cluster has an important role to facilitate affinity of enzyme to the methyl parathion substrates.