How does huperzine A enter and leave the binding gorge of acetylcholinesterase? Steered molecular dynamics simulations

The entering and leaving processes of Huperzine A (HupA) binding with the long active-site gorge of Torpedo californica acetylcholinesterase (TcAChE) have been investigated by using steered molecular dynamics simulations. The analysis of the force required along the pathway shows that it is easier for HupA to bind to the active site of AChE than to disassociate from it, which for the first time interprets at the atomic level the previous experimental result that unbinding process of HupA is much slower than its binding process to AChE. The direct hydrogen bonds, water bridges, and hydrophobic interactions were analyzed during two steered molecular dynamics (SMD) simulations. Break of the direct hydrogen bond needs a great pulling force. The steric hindrance of bottleneck might be the most important factor to produce the maximal rupture force for HupA to leave the binding site but it has a little effect on the binding process of HupA with AChE. Residue Asp72 forms a lot of water bridges with HupA leaving and entering the AChE binding gorge, acting as a clamp to take out HupA from or put HupA into the active site. The flip of the peptide bond between Gly117 and Gly118 has been detected during both the conventional MD and SMD simulations. The simulation results indicate that this flip phenomenon could be an intrinsic property of AChE and the Gly117-Gly118 peptide bond in both HupA bound and unbound AChE structures tends to adopt the native enzyme structure. At last, in a vacuum the rupture force is increased up to 1500 pN while in water solution the greatest rupture force is about 800 pN, which means water molecules in the binding gorge act as lubricant to facilitate HupA entering or leaving the binding gorge.     

Modeling the dynamics of DNA electrophoresis on a flat surface

We use molecular dynamics simulations to study the mechanism by which a flat, homogeneous surface can serve as an electrophoretic separation medium for DNA. We find that the mobility of DNA on the surface is a function of the conformation of the adsorbed DNA molecule, and that this mobility is controlled by the attraction between the DNA and the surface. Our results will provide guidelines for the fabrication of surfaces that can be used to separate DNA in a wide size range.     

氢甲酰化反应溶剂效应的量子化学研究

在B3LYP/6-31G(d, p)(Rh和P采用LANL2DZ + Polar)水平下,利用自洽反应场（SCRF）的Onsager模型,系统研究了C6H12、C6H6、THF、CH2Cl2、CH3OH和H2O（相对介电常数ε分别为2.02、2.25、7.58、8.93、32.63和78.39）六种溶剂对乙烯氢甲酰化反应中各驻点的结构和反应机理的影响.分别在上述溶剂中优化了反应物、产物、中间体和过渡态的结构,并计算了自由能和活化自由能.计算结果表明,不同溶剂对反应势能面上各驻点的结构和能量均有一定的影响,而且随着相对介电常数ε的增加,反应的活化自由能下降.水是以上六种溶剂中最佳的溶剂,这与大量的实验研究结果一致.     

Multilayer poly(vinyl alcohol)-adsorbed coating on poly(dimethylsiloxane) microfluidic chips for biopolymer separation

A poly(dimethylsiloxane) (PDMS) microfluidic chip surface was modified by multilayer-adsorbed and heat-immobilized poly(vinyl alcohol) (PVA) after oxygen plasma treatment. The reflection absorption infrared spectrum (RAIRS) showed that 88% hydrolyzed PVA adsorbed more strongly than 100% hydrolyzed one on the oxygen plasma-pretreated PDMS surface, and they all had little adsorption on original PDMS surface. Repeating the coating procedure three times was found to produce the most robust and effective coating. PVA coating converted the original PDMS surface from a hydrophobic one into a hydrophilic surface, and suppressed electroosmotic flow (EOF) in the range of pH 3-11. More than 1,000,000 plates/m and baseline resolution were obtained for separation of fluorescently labeled basic proteins (lysozyme, ribonuclease B). Fluorescently labeled acidic proteins (bovine serum albumin, beta-lactoglobulin) and fragments of dsDNA phiX174 RF/HaeIII were also separated satisfactorily in the three-layer 88% PVA-coated PDMS microchip. Good separation of basic proteins was obtained for about 70 consecutive runs.     

Sorption of 241Am by Aspergillus nigerspore and hyphae

Biosorption of ²⁴¹Am by a fungus A. niger, including the spore and hyphae, was investigated. The preliminary results showed that the adsorption of ²⁴¹Am by the microorganism was efficient. More than 96% of the total ²⁴¹Am could be removed from ²⁴¹Am solutions of 5.6-111 MBq/l (C _o) by spore and hyphaeof A. niger, with adsorbed ²⁴¹Am metal (Q) of 7.2-142.4 MBq/g biomass, and 5.2-106.5 MBq/g, respectively. The biosorption equilibrium was achieved within 1 hour and the optimum pH range was pH 1-3. No obvious effects on ²⁴¹Am adsorption by the fungus were observed at 10-45 °C, or in solutions containing Au³⁺ or Ag⁺, even 2000 times above the ²⁴¹Am concentration. The ²⁴¹Am biosorption by the fungus obeys the Freundlich adsorption equation. There was no significant difference between the adsorption behavior of A. nigerspore and hyphae. This revised version was published online in July 2006 with corrections to the Cover Date.     

A zwitterion of 1,5‐bis­(2‐hydroxy­benzamido)‐3‐aza­pentane: a two‐dimensional hydrogen‐bonding network involving dimers

The title compound, 2‐{N‐[2‐(2‐hydroxy­benzamido)ethyl­ammonio­ethyl]amino­carbon­yl}phenolate, C₁₈H₂₁N₃O₄, crystallizes in a zwitterionic form as a result of inter­molecular proton transfer and possesses a negatively charged phenolate group and a protonated amino group. The 2‐hydroxy­benzamide and 2‐(amino­carbonyl)­phenolate moieties attached to the two ends of the C—C—N—C—C backbone adopt a cis conformation in relation to this backbone. All N‐ and O‐bound H atoms are involved in hydrogen‐bond formation; the zwitterions are first linked into head‐to‐tail dimers, which are further organized into a two‐dimensional network parallel to the crystallographic bc plane.     

High-throughput semi-automated 96-well liquid/liquid extraction and liquid chromatography/mass spectrometric analysis of everolimus (RAD 001) and cyclosporin a (CsA) in whole blood

A semi-automated high-throughput liquid/liquid extraction (LLE) assay was developed for RAD001 and cyclosporin A (CsA) in human blood. After addition of internal standard and ammonium hydroxide, samples were extracted twice with methyl tert-butyl ether (MTBE). The organic extract was evaporated to dryness and reconstituted in mobile phase. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Samples were analyzed using ESI-LC/MS/MS employing the transitions of ([M + NH(4)](+) --> [M + H](+)) for CsA and ([M + NH(4)](+) --> [M + H-(CH(3)OH + H(2)O)](+)) for RAD001, under isocratic chromatographic conditions (75:25, (v/v), acetonitrile/20 mM ammonium acetate) with a run time of 3.6 min. A lower limit of quantitation (LLOQ) of 0.368 ng/mL and 5.23 ng/mL was achieved for RAD001 and CsA, respectively, using a sample volume of 0.3 mL for the analysis. The method was validated over a 3-day period and the resulting calibration curves had a correlation coefficient >0.99 over the concentration range 0.368 to 409 ng/mL and 5.24 to 1748 ng/mL for RAD001 and CsA, respectively. The inter-day coefficient of variation (CV) was less than 15% at the LLOQ for both compounds. The method was applied to the analysis of clinical samples. Under normal working conditions four 96-well plates could be extracted and LC/MS analysis completed in less than 28 h. A marked improvement in sample throughput efficiency was realized with this LLE method when compared to existing solid phase extraction (SPE) methods which deal with both RAD001 and CsA.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells.

Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931-954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each of the oncogene-transformed cell lines indicated that five major tropomyosin (Tm 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation-specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.     

Voltammetric determination of uric acid by using gold nanotubule electrode

Gold nanotubule membranes were prepared by using electroless deposition of gold within the pores and surfaces of polycarbonate track-etched membranes.And the gold nanotubule membrane was used as an electrode for determination of uric acid in urine samples for the first time.In Britton-Robinson buffer of pH 4.56,uric acid exhibited well-defined differential pulse voltammograms.And the interference between coexistent ascorbic acid and uric acid was overcome owing to the attractive ability of the gold nanotubule electrode to yield a large anodic peak difference ca.0.404 V(vs.SCE).The proposed method was then applied to the determination of uric acid in urine without any pretreatment.