Facile preparation of amperometric laccase biosensor with multifunction based on the matrix of carbon nanotubes-chitosan composite

The carbon nanotubes-chitosan (CNTs-CS) composite provides a suitable biosensing matrix due to its good conductivity, high stability, and good biocompatibility. Enzymes can be firmly incorporated into the matrix without the aid of other cross-linking reagents. The composite is easy to form insoluble film in solution above pH 6.3. Based on this, a facilely fabricated amperometric biosensor by entrapping laccase into the CNTs-CS composite film has been developed. At pH 6.0, the fungi laccase incorporated into the composite film remains better catalytic activity than that dissolved in solution. The system is in favor of the accessibility of substrate to the active site of laccase, thus the affinity to substrates is improved greatly, such as 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), catechol, and 0, with K. values of 19.86 mu M, 9.43 mu M, and 3.22 mM, respectively. The major advantages of the as-prepared biosensor are: detecting different substrates (ABTS, catechol, and 02), possessing high affinity and sensitivity, durable long-term stability, and facile preparation procedure. On the other hand, the system can be applied in fabrication of biofuel cells as the cathodic catalysts based on its good electrocatalysis for oxygen reduction.

The interaction of recombinant Pseudomonas aeruginosa exotoxin A with DNA and mimic biomembrane investigated by surface plasmon resonance and electrochemical methods

Based on the multidomain structure of Pseudomonas aeruginosa exotoxin A, a fusion protein termed rPEA has been constructed, which is expected to serve as a gene carrier in vitro. The expression and purification of rPEA are described. The basal properties of rPEA as a gene carrier are evaluated by investigating its interaction with plasmid DNA and mimic biomembrane by surface plasmon resonance (SPR) and electrochemical methods. rPEA is proved to be able to bind with plasmid DNA with high affinity. It can also interact with lipid membrane and increase permeability of the membrane, so the probe molecules can easily reach the gold surface and exhibit the electrochemical response.

Studies on the interaction of porphyrin with antibody with fluorescent spectrum and UV-Vis differential spectra

After meso-tetra (alpha, alpha, alpha, alpha-O-phenylacetyl benzene)porphyrin combined with McAb 1F2, there was a significant hyperchromic effect, indicating that the combination of porphyrin and antibody is rigid and compact, aromatic amino acids exist at the combining sites of antigen in antibody. These aromatic amino acids are Trys and Trps, but the numbers of Trp are more than that found for Trys. The stochiometric ratio of porphyrin to 1F2 is 1:1, the disassociation constant was determined as(2.084+/-0.216) x 10(-10) mol/L by a method of fluorescence quenching, showing that both have a high affinity.

Effects of La3+ and Gd3+ on Ca2+ influx in rat hepatoma H-35 cells

Effects of La3+ and Gd3+ on Ca2+ influx were investigated in rat hepatoma H-35 cells by measuring the initial rate of Ca-45(2+) uptake. It was found that the maximum initial rate of Ca2+ uptake was increased six- to ten-fold at low concentrations of La3+ and Gd3+. Kinetic analyses by measuring the initial rate of Ca2+ influx at different external Ca2+ concentrations indicated the existence of two intracellular exchangeable components in the basal Ca2+ system, with low and high affinities for Ca2+, and only one class of Ca2+ binding sites was observed in the La3+- or Gd3+-treated cells. For high affinity, La3+ and Gc(3+) increased both kinetic parameters K-m and V-max of basal Ca2+ influx. La3+ and Gd3+ compete directly with Ca2+ for Ca2+ binding site for low affinity. The kinetics is competitive.

Identification of peptides that bind with thymidylate synthase RNA using mRNA display technique

Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>10(13) different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Peptidoglycan recognition protein of Chlamys farreri (CfPGRP-S1) mediates immune defenses against bacterial infection

Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in detecting and eliminating the invading bacteria. Several PGRPs have been identified from mollusk, but their functions and the underlined mechanism are still unclear. In the present study, the mRNA expression profiles, location, and possible functions of PGRP-S1 from Zhikong scallop Chlamys farreri (CfPG RP-St) were analyzed. The CfPGRP-S1 protein located in the mantle, gill, kidney and gonad of the scallops. Its mRNA expression in hemocytes was up-regulated extremely after PGN stimulation (P < 0.01), while moderately after the stimulations of LPS (P < 0.01) and beta-glucan (P < 0.05). The recombinant protein of CfPGRP-S1 (designated as rCfPGRP-S1) exhibited high affinity to PGN and moderate affinity to LPS, but it did not bind beta-glucan. Meanwhile, rCfPGRP-S1 also exhibited strong agglutination activity to Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and weak activity to Gram-negative bacteria Escherichia coli. More importantly, rCfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn2+. These results indicated that CfPGRP-S1 could not only serve as a pattern recognition receptor recognizing bacterial PGN and LPS, but also function as a scavenger involved in eliminating response against the invaders. (C) 2010 Elsevier Ltd. All rights reserved.

胸苷酸合成酶和二氢叶酸还原酶mRNA亲和肽的发现及mRNA体外展示技术的优化

胸苷酸合成酶（thymidylate synthase，简称TS）和二氢叶酸还原酶（dihydrofolate reductase, 简称DHFR）都是叶酸依赖性酶，在维持DNA合成和DNA修复上发挥关键作用，并且多年来一直是肿瘤研究和化疗的重要靶点。我们前期的研究发现，TS和DHFR在翻译水平上存在负反馈调控机制。人TS和DHFR可以与其自身的mRNA结合，从而抑制mRNA的表达，化疗药物可以与TS或者DHFR相互作用，形成的复合物不能与TS mRNA结合， 使负反馈机制丧失。因此深入研究TS和DHFR的翻译调控机理，对阐明肿瘤抗药性机制，对发现新的抗肿瘤药物和肿瘤的治疗都具有十分重要的意义。 本论文利用mRNA体外展示技术，构建多肽库（约10万亿种多肽分子），利用多种实验手段将mRNA体外展示技术进行优化，提高了多肽库融合肽的产量，提高了mRNA体外展示技术筛选的特异性。将TS mRNA分子上的顺式因子TS30 RNA固定于磁珠上，将融合肽库与顺式因子作用，经过6轮循环，由多肽库中获得了与TS mRNA高度亲和的多肽序列，体外结合实验证明亲和肽可以与TS全长mRNA结合，体外翻译实验证明多肽可以抑制TS mRNA的翻译。并且利用phage display技术由噬菌体肽库（12个氨基酸随机肽库）经过四轮筛选，分别筛选到TS和DHFR的亲和肽，凝胶阻滞实验证明它们分别能与TS和DHFR mRNA结合。 本论文利用的展示技术可以广泛应用于特异靶点的蛋白质筛选，并且本论文筛选到的TS和DHFR亲和肽可以作为TS和DHFR的抑制剂，从而为获得新型的抗肿瘤药物奠定基础。

Organochlorine micropollutants in the Jiulong River Estuary and Western Xiamen Sea, China

Organochlorine contaminants including 12 polychlorinated biphenyl (PCB) congeners and 18 insecticides were determined in water, pore water and sediments of the Jiulong River Estuary and Western Xiamen Sea, China. The results showed that the levels of the total PCBs ranged from non-detectable to 1500 ngl(-1) in water, from 209 to 3870 ngl(-1) in pore water, and from 2.78 to 14.8 ng g(-1) dry weight in sediments. Total organochlorine insecticide concentrations were from below the limit of detection to 2480 ngl(-1) in water, from 267 to 33400 ngl(-1) in pore water, and from 4.22 to 46.3 ng g(-1) dry weight in sediments. Concentrations of PCBs and insecticides in pore water were significantly higher than those in surface water, due to the high affinity of these hydrophobic compounds for sediment phase. The PCB congeners with the highest concentrations were CB153, CB180 and CB194, which together accounted for 68-87% of total PCBs in water, pore water and sediment. Among the hexachlorocyclohexane (HCH) compounds, beta-HCH was found to be a major isomer. Analysis of 1,1,1-trichloro-2,2-bis-chlorophenyl-ethane (DDT) and its metabolites showed that 1, 1-dichloro-2[o-chlorophenyl]-2[p-chlorophenyl]-ethylene (DDE) was dominant in the group. In comparison to a 1998 study in the Western Xiamen Sea, levels of organochlorines were enhanced due probably to recent inputs and changes in sediments. (c) 2004 Elsevier Ltd. All rights reserved.

Phenylurea herbicides-selective polymer prepared by molecular imprinting using N-(4-isopropylphenyl)-N'-butyleneurea as dummy template

A phenylurea herbicides-selective molecularly imprinted polymer (MIP) was prepared using N-(4-isopropylphenyl)-N'-butyleneurea as a dummy template and toluene as a porogen. The experimental results showed that the optimum molar ratio of template, functional monomer (MAA) and cross-linker (EDMA) was 1:8:20. Scatchard analysis showed that two classes of binding sites were formed in the imprinted polymer with dissociation constants of 26.81 mu L mol l(-1) and 1.428 mmol l(-1). The affinity and selectivity of MIP for phenylurea herbicides were studied. Among the 14 phenylurea herbicides tested, the MIP prepared showed obviously high affinity and selectivity for 10 chemicals (monuron, diuron, isoproturon, fenuron, chlortoluron, difenoxuron, metoxuron, neburon, buturon and fluometuron) with dichloromethane containing 10% hexane as mobile phase while non-imprinted polymer showed very low affinity for all the phenylurea herbicides tested. The experimental and calculated results also indicated that the size and property of the group at the N' position of phenylurea molecules have great influence on the affinity of MIP for them and the recognition site is mainly located at the N' position of phenylurea herbicides. (c) 2005 Elsevier B.V. All rights reserved.

Shock Tube Studies on the Nonequlibrium Electron Affinity Kinetics of Fluorine Atom at High Temperatures

To study electron affinity kinetics, a shock tube method was applied, in which the test gas was ionized by a reflected shock wave and subsequently quenched by a strong rarefaction wave. As the quenching speed of 106 K/s was reached, a nonequilibrium ionization-recombination process occurred, which was dominated by ion recombination with electrons. A Langmuir electrostatic probe was used to monitor variation in the ion number density at the reflection shock region. The working state of the probe was analyzed...

High-Integrated-Photosensitivity Negative-Electron-Affinity GaAs Photocathodes with Multilayer Be-Doping Structures

The effect of changing Be doping concentration in GaAs layer on the integrated photosensitivity for nega- tive-electron-affinity GaAs photocathodes is investigated. Two GaAs samples with the monolayer structure and the muhilayer structure are grown by molecular beam epitaxy. The former has a constant Be concentration of 1 × 10^19 cm^-3, while the latter includes four layers with Be doping concentrations of 1 × 10^19, 7 × 10^18, 4 × 10^18, and 1 × 10^18 cm^-3 from the bottom to the surface. Negative-electron-affinity GaAs photocathodes are fabricated by exciting the sample surfaces with alternating input of Cs and O in the high vacuum system. The spectral response results measured by the on-line spectral response measurement system show that the integrated photosensitivity of the photocathode with the muhilayer structure enhanced by at least 50% as compared to that of the monolayer structure. This attributes to the improvement in the crystal quality and the increase in the surface escape probability. Different stress situations are observed on GaAs samples with monolayer structure and muhilayer structure, respectively.