Phosphate binding protein as the biorecognition element in a biosensor for phosphate

This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins.     

Early intervention for spelling problems: Teaching functional spelling units of varying size with a multiple-connections framework.

Poor spellers in 2nd grade (n?=?128) participated in 24 20-min sessions that included (a) direct instruction in the alphabet principle (most frequent phoneme-spelling connections); (b) modeling of different approaches, singly and in combination, for developing connections between spoken and written words for 48 words ordered by sound-spelling predictability; and (c) practice in composing. Results of this multilayered intervention showed that (a) more than 1 way of developing sound-spelling connections is effective in teaching spelling but that after training in the alphabet principle, combining whole word and onset-rime training is most effective in achieving transfer of the alphabet principle across word contexts; (b) functional spelling units of not only a single letter but also 2 or more letters are important in beginning spelling; and (c) training in spelling transfers to composition and word recognition. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evaluation of Corn/Soybeans Separability Using Thematic Mapper And Thematic Mapper Simulator Data

Multitemporal Thematic Mapper, Thematic Mapper Simulator, and detailed ground truth data were collected for a 9-by 1-km sample segment in Webster County, Iowa, in the summer of 1982. Three dates were acquired each with Thematic Mapper Simulator (June 7, June 23, and July 31) and Thematic Mapper (August 2, September 3, and October 21). The Thematic Mapper Simulator data were converted to equivalent TM count values using TM and TMS calibration data and model based estimates of atmospheric effects. The July 31, TMS image was compared to the August 2, TM image to verify the conversion process. A quantitative measure of proportion estimation variance (Fisher information) was used to evaluate the corn/soybeans separability for each TM band as a function of time during the growing season. The additional bands in the middle infrared allowed corn and soybeans to be separated much earlier than was possible with the visible and near-infrared bands alone. Using the TM and TMS data, temporal profiles of the TM principal components were developed. The greenness and brightness exhibited behavior similar to MSS greenness and brightness for corn and soybeans.     

Monitoring ligand modulation of protein-protein interactions by mass spectrometry: estrogen receptor alpha-SRC1

Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.     

Effect of Surface-Active Substances on the Rheological Properties of Silicon Carbide Suspensions in Paraffin

In this paper, the possibility of stabilization of SiC suspensions in paraffin (hot-molding slips) was investigated from the colloid science point of view. By considering the surface properties of finely dispersed silicon carbide, surface-active agents (fatty amine and alkylsuccinimide) which are suitable for surface modification by chemical adsorption on SiC were selected. Adsorption of fatty amines and alkylsuccinimide was carried out in model experiments in chloroform. Increasing the length of the carbon–hydrogen chain caused the adsorption to decrease, and the adsorption area of one molecule in the saturated adsorption layer to increase. The stabilizing effect of surface-active agents resulted in improved flow behavior of hot-molding slips. The efficiency of surface-active agents increased with increased adsorption layer thickness. In highly concentrated SiC hot-molding slips, a combination of alkylsuccinimide and fatty amine proved to be very efficient. This synergistic effect of long- and short-chained surface-active agents is discussed in terms of an adsorption layer model.     

Electrophoretic deposition for fabrication of ceramic microparts

Electrophoretic deposition (EPD) is mostly used for producing moulds and layers. The present work shows the fabrication of ceramic microparts by the use of structured electrodes. As electrode structures euro coins and spinning nozzles were used. Additionally a cost effective and simple method was developed, which allows preserving the original master mould by using microstructured silicone moulds as substrate. This was enabled by coating the silicone moulds with graphite to obtain an electrically conductive surface, required for electrophoretic deposition.     

800 kV叠装式瓷绝缘子大雨下的性能评估

An 800 kV electric power substation can have more than one hundred of porcelain multicone type insulators supporting busbar structures.Two or more sections in series,having middle and end fittings,compose these insulators.Ageing process can cause degradation of the cement used in this type of insulator and this fact can affect its dielectric performance under heavy rain.This paper presents results of an investigation based on power frequency high voltage tests performed on 800 kV porcelain multicone type insulators,removed from service after having operated for more than 20 years,as well a non-used one that had been stored on site for long time.The insulators were tested in different arrangements:each section at a time and the two sections assembled in series,as actually used insulator columns.The tests were carried out under artificial rain ranging from 1 mm/min to 5 mm/min.The results have confirmed a reduction of up to 30% in the insulator power frequency flashover voltage under 5 mm/min rain conditions and gave important information to the utility about radial cracks that were observed in many insulators and about the discharge mechanisms along the insulators under rain.     

Effect of Dietary Cholesterol and Fat on Cell Cholesterol Transfer to Postprandial Plasma in Hyperlipidemic Men

Postprandial chylomicrons are potent ultimate acceptors of cell membrane cholesterol and are believed to accelerate reverse cholesterol transport (RCT). We compared the effects of meals rich in polyunsaturated fat (PUFA) and either high (605 mg) or low (151 mg) in cholesterol and a meal rich in dairy fat (DF) in the form of cream on net in vitro transport of red blood cell (RBC) membrane cholesterol to 4 and 6 h postprandial plasma in eight normotriglyceridemic (NTG-H) and eight hypertriglyceridemic (HTG-H) men with mild to moderate hypercholesterolemia. In HTG-H men, cell cholesterol accumulation in 6-h postprandial plasma was significantly (P = 0.02) less after the PUFA-HC meal compared with the other meals. The significant (P < 0.001) increase in cell plus endogenous cholesterol accumulation in the triglyceride-rich lipoprotein (TRL) fraction of 4 h postprandial plasma incubated with RBC was significantly (P = 0.007) higher after the PUFA-HC meal compared with DF meal in HTG-H men. In NTG-H men, cholesterol accumulation in plasma and plasma lipoproteins in the presence and absence of RBC was not significantly affected by the type of meal ingested. These data suggest that addition of large amounts of cholesterol to a PUFA meal may impair diffusion-mediated transport of cell membrane cholesterol to postprandial plasma and that replacing DF with PUFA in a meal increases postprandial lipemia and may potentially increase cholesterol accumulation in atherogenic postprandial TRL in HTG-H men.