A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor

The residues in the second extracellular loop (eLP2) of the prostanoid receptors, which are important for specific ligand recognition, were previously predicted in our earlier studies of the thromboxane A2 receptor (TP) using a combination of NMR spectroscopy and recombinant protein approaches. To further test this hypothesis, another prostanoid receptor, the prostacyclin receptor (IP), which has opposite biological characteristics to that of TP, was used as a model for these studies. A set of recombinant human IPs with site-directed mutations at the nonconserved eLP2 residues were constructed using an Ala-scanning approach, and then expressed in HEK293 and COS-7 cells. The expression levels of the recombinant receptors were six-fold higher in HEK293 cells than in COS-7 cells. The residues important for ligand recognition and binding within the N-terminal segment (G159, Q162, and C165) and the C-terminal segment (L172, R173, M174, and P179) of IP eLP2 were identified by mutagenesis analyses. The molecular mechanisms for the specific ligand recognition of IP were further demonstrated by specific site-directed mutagenesis using different amino acid residues with unique chemical properties for the key residues Q162, L172, R173, and M174. A comparison with the corresponding functional residues identified in TP eLP2 revealed that three (Q162, R173, and M174) of the four residues are nonconserved, and these are proposed to be involved in specific ligand recognition. We discuss the importance of G159 and P179 in ligand recognition through configuration of the loop conformation is discussed. These studies have further indicated that characterization of the residues in the eLP2 regions for all eight prostanoid receptors could be an effective approach for uncovering the molecular mechanisms of the ligand selectivities of the G-protein-coupled receptors.     

Egg white, a favourable solvent to extract the active ingredient of Mylabris phalerata

To characterize the role of hen eggs used for analyzing the insect Mylabris phalerata a traditional Chinese medicine for curing liver cancer, egg white was separated from the whole egg and added to M. phalerata. The volatile compounds found in egg white when added to M. phalerata and therefore in M. phalerata itself were analyzed by GC. Cantharidin was detected both in egg white added to M. phalerata and M. phalerata itself. Egg white can be used as a favourable solvent for extracting the active ingredient of M. phalerata.     

The use of BeWo cells as an in vitro model for placental iron transport

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 +/- 0.5% of the total dose was transported after 8 h, equivalent to 38.8 +/- 2.1 pmol.cm(-2).h(-1). Transfer of Tf across the cell layer was much more limited; 2.4 +/- 0.2% of the total dose was transported after 8 h, equivalent to 5.0 +/- 0.4 pmol.cm(-2).h(-1). Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.     

Responses of oxidation rate and microbial communities to methane in simulated landfill cover soil microcosms

CH4 oxidation capacities and microbial community structures developed in response to the presence of CH4 were investigated in two types of landfill cover soil microcosms, waste soil (fine material in stabilized waste) and clay soil. CH4 emission fluxes were lower in the waste soil cover over the course of the experiment. After exposure to CH4 flow for 120 days, the waste soil developed CH4 oxidation capacity from 0.53 to 11.25-13.48micromol CH4gd.w.(-1)h(-1), which was ten times higher than the clay soil. The topsoils of the two soil covers were observed dried and inhibited CH4 oxidation. The maximum CH4 oxidation rate occurred at the depth of 10-20cm in the waste soil cover (the middle layer), whereas it took place mainly at the depth of 20-30cm in the clay soil cover (the bottom layer). The amounts of the phospholipid fatty acid (PLFA) biomarks 16:1omega8c and 18:1omega8c for type I and II methanotrophs, respectively, showed that type I methanotrophic bacteria predominated in the clay soil, while the type II methanotrophic bacteria were abundant in the waste soil, and the highest population in the middle layer. The results also indicated that a greater active methanotrophic community was developed in the waste soil relative to the clay soil.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.     

Soya food intake and risk of endometrial cancer among Chinese women in Shanghai: population based case-control study

Objective To evaluate the association of intake of soya food, a rich source of phytoestrogens, with the risk of endometrial cancer.Design Population based case-control study, with detailed information on usual soya food intake over the past five years collected by face to face interview using a food frequency questionnaire.Setting Urban Shanghai, China.Participants 832 incident cases of endometrial cancer in women aged of 30 to 69 years diagnosed during 1997-2001 and identified from the Shanghai Cancer Registry; 846 control women frequency matched to cases on age and randomly selected from the Shanghai Residential Registry.Main outcome measures Odds ratios for risk of endometrial cancer in women with different intakes of soya foods.Results Regular consumption of soya foods, measured as amount of either soya protein or soya isoflavones, was inversely associated with the risk of endometrial cancer. Compared with women with the lowest quarter of intake, the adjusted odds ratio of endometrial cancer was reduced from 0.93 to 0.85 and 0.67 with increasing quarter of soya protein intake (P for trend 0.01). A similar inverse association was observed for soya isoflavones and soya fibre intake. The inverse association seemed to be more pronounced among women with high body mass index and waist:hip ratio.Conclusion Regular intake of soya foods is associated with a reduced risk of endometrial cancer.     

Effects of litter incorporation and nitrogen fertilization on the contents of extractable aluminium in the rhizosphere soil of tea plant (Camallia sinensis (L.) O. Kuntze)

The effects of litter incorporation and nitrogen application on the properties of rhizosphere and bulk soils of tea plants (Camellia sinensis (L.) O. Kuntze) were examined in a pot experiment. Total of 8 treatments included four levels of tea litter additions at 0, 4.9, 9.8, and 24.5 g kg^–1 in combination with two N levels (154.6 mg kg^–1 and without). After 18 months of growth the rhizosphere soil was collected by removing the soil adhering to plant roots and other soil was referred to as bulk soil. The dry matter productions of tea plants were significantly increased by N fertilization and litter incorporation. The effect of litter was time-depending and significantly decreased the content of exchangeable Al (Al_ex, by 1 mol L^–1 KCl) and Al saturation at 9 months after litter incorporation whereas soil pH was not affected, although the litter contained high Al content. After 18 months, the contents of extractable Al by dilute CaCl₂, CuCl₂ + KCl, NH₄OAC, ammonium oxalate and sodium citrate (Al_CaCl2, Al_Cu/KCl, Al_NH4OAC, Al_Oxal, and Al_Cit respectively) and Al_ex, were not affected by litter application, except that of Al_CaCl2 in the rhizosphere soil which was decreased following litter additions. Nitrogen fertilization with NH₄ ⁺ (urea and (NH₄)₂SO₄) significantly reduced soil pH, the contents of exchangeable Ca, K, Mg and base saturation while raised extractable Al levels (Al_CaCl2, Al_Cu/KCl, Al_NH4OAC, and Al_ex). In the rhizosphere soils exchangeable K accumulated in all treatments while exchangeable Ca and Mg depleted in treatments without litter application. The depletions of Ca and Mg were no longer observed following litter incorporation. This change of distribution gradients in rhizosphere was possibly due to the increase of nutrient supplies from litter decomposition and/or preferable root growth in soil microsites rich in organic matter. Lower pH and higher extractable Al (Al_CaCl2, Al_ex, and Al_NH4OAC) in the rhizosphere soils, regardless of N and litter treatments, were distinct and consistent in all treatments. Such enrichments of extractable Al in the rhizosphere soil might be of importance for tea plants capable of taking up large amounts of Al.     

苏云金芽胞杆菌产苏云金素菌株CT-43及其 缺失突变株的差异蛋白

[目的]苏云金素(Thuringiensin)的合成和代谢途径的相关研究在国内外一直进展缓慢,本文拟从蛋白质组水平揭示与苏云金素合成或代谢相关的蛋白.[方法]利用双向电泳技术研究了高产苏云金素的苏云金芽胞杆菌野生菌株CT-43、其高产突变菌株CT-43-1C及不产突变菌株BMB0806在蛋白表达水平的差异,然后对差异蛋白点进行质谱鉴定,最后对鉴定出的蛋白进行生物信息学分析.[结果]与野生型和高产菌株相比,在BMB0806中发现了13个差异显著的蛋白点,鉴定出了其中的9个,生物信息学结果显示有6个蛋白可能与苏云金素合成或代谢相关.[结论]通过蛋白质组研究找到了6个可能与苏云金素合成或代谢相关的蛋白,为苏云金素合成基因簇的克隆和合成途径的验证提供了有力的证据.     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.