SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Two methods are described for adenovirus capsid disruption and extraction of acid-soluble proteins from the viral core. The acid-soluble material of adenovirus consisted of three major proteins, one of them being selectively extracted after mild disruption of the virus particle. Some chemical properties of these proteins are reported.
In LC/MS, nonpolar substances in the majority of cases cannot be ionized by standard electrospray ionization (ESI) because they obviously lack a site for protonation or deprotonation. The ionization of carotenoids and tocopherols can be greatly enhanced by the addition of silver ions. The Ag(+)-carotenoid and Ag(+)-tocopherol adducts thus formed render these substances amenable to MS. alpha-, beta-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, and the various isomers of lycopene and beta-carotene were separated by C30 RP-HPLC and could be identified by online ESI-MS. A mixture of six different carotenoids was analyzed by scanning the mass range from m/z 500 to 800. The mass spectra of the peaks revealed that all carotenoids and most tocopherols were partially oxidized to radical cations. The detection limit for canthaxanthin was approximately 500 fmol while that of beta-carotene was below 300 fmol. An increase in sensitivity in the MRM mode can be attained by monitoring ions formed by loss of elemental silver from the adducts in the CID cell. Dichloromethane extracts of tomato, carrot, and vegetable juices, a vitamin drink, and a commercial infant food product were analyzed by LC/MS. After postcolumn argentation, from the mass-selective extracts of the TIC, the carotenoids and tocopherols present could be identified by their masses and their retention times. For all studies, a silver perchlorate solution with an overall concentration of 50 micrograms/mL was used.
A human leukaemia cell line--HL-60--can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.
In this work, the analysis of a group of four fungicides (pyrimethanil, nuarimol, procymidone and cyprodinil) and one insecticide (pirimicarb) by micellar electrokinetic chromatography (MEKC) with UV detection using the on-line preconcentration strategy called reversed electrode polarity stacking mode (REPSM) is proposed. After optimisation, an adequate separation electrolyte for the separation and stacking of these pesticides was obtained which consisted of 100 mM borate, 60 mM sodium dodecyl sulphate (SDS), at pH 9.0 and 2% 2-propanol. The use of this running buffer together with the REPSM preconcentration method provided limits of detection (LODs) between 38.3 and 241 microg/L. In order to apply the developed methodology for the analysis of these pesticides in wine samples, several off-line preconcentration strategies (mainly, solid-phase extraction, SPE, and solid-phase microextraction, SPME) were tested. Although the use of a SPE procedure, optimized in this work for water samples, using Oasis HLB cartridges, provided mean recovery values between 79 and 100% for spiked water samples, it could not be applied to the extraction of these pesticides from wine samples due to high interference from the sample matrix. However, the use of a SPME procedure using polydimethylsiloxane/divynilbenzene (PDMS/DVB) fibers allowed the selective extraction of four of the five pesticides which could be perfectly determined. The final combination of the off-line SPME and on-line REPSM preconcentration strategies allowed obtaining LODs between 17.6 and 32.3 microg/L.
A zwitterionic bis(guanidinium) ionophore bearing an anionic closo-borane cluster (1) and a dihydrochloride analogue (2) are investigated in polymeric membrane ion-selective electrodes (ISEs). Both compounds have been previously shown to complex and selectively extract oxoanions. By systematic variation of the kind and concentration of the ion-exchanger sites in the membrane, the optimal performance with the so far best sulfate selectivity is found for ISE membranes based on the dihydrochloride, whereas those with the zwitterion analogue are shown to possess a reasonably good selectivity for monohydrogen phosphate.
A series of novel N-chromogenic calix[4]arene azacrown ethers were synthesized as selective extractants of potassium ion. 1,3-Alternate calix[4]arene azacrown ethers were prepared by reacting 25,27-dipropyloxy-26,28-bis(5-chloro-3-oxapentyloxy) calix[4]arenes with p-toluenesulfonamide in the presence of potassium carbonate. The coupling reaction of calix[4]arene azacrown ether with 2-hydroxy-5-nitrobenzyl bromide in the presence of triethylamine in THF gave the chromogenic calix[4]arene azacrown ether in moderate yield. These compounds show high potassium selectivity over other metal ions as shown by two-phase extraction, bulk liquid membrane, and 1H NMR studies on a ligand-metal complex. It is assumed that the OH of the chromogenic group attached on nitrogen can assist the complexation by encapsulation of the metal.
The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.
This assay for phylloquinone (vitamin K1) in plasma requires a single liquid-chromatographic step. Much smaller volumes of plasma (0.5-1.0 mL) are required than in previous assays. Before liquid chromatography, we purified crude lipid extracts by conventional chromatography on silica, then extracted the lipid fraction by dissolving it in an acidic mixture of hexane/acetonitrile (1/4 by vol) containing 70 mmol of zinc chloride per liter. The vitamin K1 was selectively extracted into acetonitrile after being converted to vitamin K1 hydroquinone by addition of zinc metal. This procedure removes greater than 99% of contaminating lipids. We injected the lipid extract directly onto a reversed-phase column after re-converting the vitamin K1 hydroquinone to vitamin K1. Vitamin K1 was quantified by comparison with the internal standard (dihydro-vitamin K1) and detected fluorometrically after post-column "on-line" reduction to the hydroquinone with zinc metal. The lower limit of detection for vitamin K1 in the final reversed-phase system was about 0.05 microgram/L plasma; CVs for replicates were less than 10%. The mean concentration of vitamin K1 in plasma from 22 healthy fasting adults was 0.55 (range 0.09-2.12) micrograms/L.
Circulating ethyl loflazepate metabolites in the baboon were determined, following a single oral administration of the 14C-labelled drug, by radio-high-performance liquid chromatography with injection of crude plasma samples and by selective extraction with thin-layer chromatographic analysis of the radioactive components. Metabolites identified by comparing their chromatographic behaviour with synthetic standards were loflazepate, descarboxyloflazepate and 3-hydroxydescarboxyloflazepate. Loflazepate represented about 70% of the circulating radioactivity; the two other metabolites were present in amounts too small to allow accurate quantification. The parent drug was not present in the blood. Comparison of high-performance liquid chromatography with solvent extraction demonstrated the inaccuracy of the latter to be caused by the conversion of loflazepate to descarboxyloflazepate.
Artificial chromogenic substrates, derived from 2-(N-hexadecanoylamino)-4-nitrophenol, can be used to assay sphingomyelin phosphodiesterase, glucosylceramidase, or galactosylceramidase. Nevertheless, these enzymatic spectrophotometric assays cannot be realized on tissue preparations containing hemoglobin which interferes in the measurement. We present a selective extraction method of 2-(N-hexadecanoylamino)-4-nitrophenol which allows to avoid hemoglobin interference in this spectrophotometric assay of 2-(N-hexadecanoylamino)-4-nitrophenol. The solvents used have been tested to obtain on the one hand maximal absorbance and organic extraction of 2-(N-hexadecanoylamino)-4-nitrophenol, and on the other hand the minimal hemoglobin interference. None of the pure solvents studied being suitable, two solvent mixtures were selected: ethyl acetate/2-propanol (5/1) and 2-ethyl-1-hexanol/4-methyl-2-pentanone (1/1). These methods were tested to determine sphingomyelinase activity in enzymatic preparations and prove that they are available for lysosomal hydrolase assays using these colorimetric substrates.
BACKGROUND: Local nasal immunotherapy (LNIT) is an effective and safe alternative to conventional subcutaneous immunotherapy. A specific nasal provocation test (SNPT) could be used to indicate the optimal subclinical dose to carry out LNIT. OBJECTIVE: We hypothesize that LNIT could be carried out with only one predefined dose for all patients, so we have evaluated the efficacy and the tolerability of LNIT administered at steady dosages in patients with seasonal allergic rhinitis. METHODS: Twenty grass pollen-sensitized patients suffering from seasonal allergic rhinitis were studied in a randomized, double-blind, placebo-controlled trial. The treatment was carried out according to a schedule based on the administration of a steady subclinical dosage of the allergenic extract, selected on the basis of the sensitivity threshold of the SNPT. The patients were divided into two groups of 10 people each, which were treated either with grass pollen extract in a hydroglyceric solution or with placebo. RESULTS: During the peak pollen period, with regard to the placebo (P) group, in the grass treated (GT) group a significant decrease of both nasal symptoms (P = .021) and consumption of antihistamines (P = 0.047) was found. Furthermore, only in the GT group was the provocative dose assessed by the SNPT significantly lower (P = .049) at the end of the treatment. In this group of patients an inverse correlation between such provocative dose and the nasal symptom score reported during the peak pollen period was also evidenced (r = 0.708; P = .038). Adverse reactions to LNIT were mild, rare, and did not interfere with the completion of the therapeutic schedule. CONCLUSIONS: Our study indicates that LNIT, when administered at steady dosages, may be proposed as a treatment for grass pollen seasonal allergic rhinitis as it appears to be effective and well tolerated.
Aqueous extracts of ten Chinese herbs were evaluated for their radical scavenging activity by a GC-MS method based on the Fenton reaction system. Hydroxylation of salicylate and phenylalanine is widely used as an index of hydroxyl radical formation in vivo and in vitro. A problem associated with quantifying product from such reactions is the generation of complex reaction products that increase background 'noise' and reduce sensitivity for the target product. The aim of this investigation was to develop a GC-MS methodology to assess in vitro hydroxyl radical production. In this method, hydroxyl radical was trapped by p-hydroxyphenylacetic acid to form 3,4-dihydroxyphenylacetic acid (DOPAC) which was then selectively extracted from the reaction mixture using aluminium oxide and assayed by GC-MS. Selective adsorption and desorption of the catechol nucleus from aluminium oxide was shown to eliminate interference from non-catechol reaction products effectively. This system was applied to examine the hydroxyl radical scavenging activity of different herbal extracts. The results showed that the herb Dimocaepus Longan Lour exhibited the highest radical scavenging activity of all the herbs examined. With the use of a stable isotope-labelled internal standard, this system could be readily applied to in vitro methods which use 4-hydroxybenzoic acid as a substrate for the hydroxyl radical.
A venom sac extract selectively and irreversibly suppressed excitatory postsynaptic potentials in the lobster neuromuscular junction without affecting inhibitory postsynaptic potentials. Glutamate-induced depolarization of the postsynaptic membrane was blocked by the venom extract, whereas membrane depolarization by aspartate was unaffected.
A simple and rapid procedure for the assay of acyl-coenzyme A:1-acyl-sn-glycero-3-phosphocholine acyltransferase (lysolecithin acyltransferase, LLAT [EC 2.3.1.23]) activity in crude enzyme preparations is described. The incubation system utilizes lysolecithin and [1-14C]-oleoyl-coenzyme A as substrates. Labeled fatty acid released due to accompanying acyl-coenzyme A hydrolase [EC 3.1.2.2]activity is first removed by di-isopropyl ether extraction. The labeled lecithin produced due to LLAT action is then quantitatively recovered by partition of the incubation medium with di-isopropyl ether-n-butanol 60:40 (v/v). Selective extraction of the labeled lecithin formed and avoidance of customary thin-layer chromatographic isolation procedures permits assay of LLAT activity with excellent accuracy at a substantial saving of time. The entire assay can be completed in less than 30 min as compared to 2-3 hrs when following conventional procedures.
The AOAC official first action method for methyl mercury in fish and shellfish was modified to provide more rapid determination. Methyl mercury is isolated from homogenized, acetone-washed tissue by addition of HCl and extraction by toluene of the methyl mercuric chloride produced. The extract is analyzed by electron capture gas chromatography (GC) on 5% DEGS-PS treated with mercuric chloride solution. The quantitation limit of the method is 0.25 micrograms Hg/g. Swordfish, shark, tuna, shrimp, clams, oysters, and NBS Research Material-50 (tuna) were analyzed for methyl mercury by the AOAC official first action method. All products also were analyzed by the modified method and the AOAC official method for total Hg. In addition, selected extracts obtained with the modified method were analyzed by GC with Hg-selective, microwave-induced helium plasma detection. There was no significant difference between the results for the various methods. Essentially all the Hg present (determined as total Hg) was in the organic form. Coefficients of variation from analyses by the modified method ranged from 1 to 7% for fish and shellfish containing methyl mercury at levels of 0.50-2.30 micrograms Hg/g. The overall average recovery was 100.5%.
Proanthocyanidins are supposed to have some therapeutical properties as antioxidants and antineoplasics. Most of the proanthocyanidins, however, are not commercialized since their separation from natural sources is either very expensive or not well-known. In this work, the feasibility of application of mixtures of carbon dioxide and alcohol under supercritical conditions for selective extraction of some phenolic compounds from grape seeds has been studied, among them some low polymerized proanthocyanidins, their main monomer units, (+)-catechin and (-)-epicatechin, and some low molecular weight phenolic compounds, like gallic acid. An analytical-scale supercritical fluid extractor, whose operation was previously optimized, was used to carry out the experiments. A commercial concentrate of complex phenols and tannins from grape seeds was subjected to supercritical extraction in order to find the best operation conditions before directly extracting defatted milled grape seeds. The solvent capacity was found to increase with pressure and with the amount of alcohol used as cosolvent as expected. Such variation in solvent capacity could be used for design of a selective separation process where individual phenolic compounds or groups of them could be obtained. HPLC coupled with two types of detectors, diode array and mass spectrometry, was used for tentative identification and quantification of complex phenols and tannins in the extracts and in the raw materials used for extraction.
Current methods of four-dimensional (4D) CT imaging of the thorax synchronise the acquisition with a respiratory signal to restrospectively sort acquired data. The quality of the 4D images relies on an accurate description of the position of the thorax in the respiratory cycle by the respiratory signal. Most of the methods used an external device for acquiring the respiratory signal. We propose to extract it directly from thorax cone-beam (CB) CT projections. This study implied two main steps: the simulation of a set of CBCT projections, and the extraction, selection and integration of motion information from the simulation output to obtain the respiratory signal. A real respiratory signal was used for simulating the CB acquisition of a breathing patient. We extracted from CB images a respiratory signal with 96.4% linear correlation with the reference signal, but we showed that other measures of the quality of the extracted respiratory signal were required.
Caffeine is a popular compound for phenotyping individuals for CYP4501A2, xanthine oxidase (XO) and N-acetyltransferase (NAT) utilising urinary metabolites. The analysis is complex since at least thirteen metabolites are excreted by man. Past methods have been less than satisfactory in that either not all the metabolites have been resolved and/or extractions selective for particular groups of metabolites are required prior to chromatography. We report a method for the rapid analysis of caffeine and metabolites in urine that negates the requirement for an extraction step, and also a method for plasma analysis.