Search results for " fractionation"
showing 10 items of 212 documents
Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle.
2003
From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of approximately 12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change…
Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites.
1999
Abstract Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by …
Translocation of cdk2 to the nucleus during G1-phase in PDGF-stimulated human fibroblasts.
1997
We studied the subcellular distribution of cdk2 in synchronized, PDGF-stimulated human fibroblasts (FH109). After contact inhibition and serum depletion, more than 95% of FH109 cells were arrested in G0/G1-phase. PDGF-AB led to a 16-fold increase in proliferation compared with untreated cells. Cell cycle progression was studied by flow cytometric analysis, [3H]thymidine incorporation, and phosphorylation of the retinoblastoma gene product, pRB. Using Western blot analysis after subcellular fractionation, we revealed that after PDGF stimulation the phosphorylated (Thr 160), i.e., activated, form of cdk2 (33 kDa) first appeared in the nucleus at late G1-phase and persisted throughout until to…
Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila’s Whole Larvae and Tissues
2015
The identification and study of protein’s function in several model organisms is carried out using both nuclear and cytoplasmic extracts. For a long time, Drosophila’s embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood. Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila’s larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.
UVA irradiation induces relocalisation of the DNA repair protein hOGG1 to nuclear speckles
2006
The DNA glycosylase hOGG1 initiates base excision repair (BER) of oxidised purines in cellular DNA. Using confocal microscopy and biochemical cell fractionation experiments we show that, upon UVA irradiation of human cells, hOGG1 is recruited from a soluble nucleoplasmic localisation to the nuclear matrix. More specifically, after irradiation, hOGG1 forms foci colocalising with the nuclear speckles, organelles that are interspersed between chromatin domains and that have been associated with transcription and RNA-splicing processes. The use of mutant forms of hOGG1 unable to bind the substrate showed that relocalisation of hOGG1 does not depend on the recognition of the DNA lesion by the en…
Influence of15N enrichment on the net isotopic fractionation factor during the reduction of nitrate to nitrous oxide in soil
2007
5 pages; International audience; Nitrous oxide, a greenhouse gas, is mainly emitted from soils during the denitrification process. Nitrogen stable-isotope investigations can help to characterise the N(2)O source and N(2)O production mechanisms. The stable-isotope approach is increasingly used with (15)N natural abundance or relatively low (15)N enrichment levels and requires a good knowledge of the isotopic fractionation effect inherent to this biological mechanism. This paper reports the measurement of the net and instantaneous isotopic fractionation factor (alpha(s/p) (i)) during the denitrification of NO(3) (-) to N(2)O over a range of (15)N substrate enrichments (0.37 to 1.00 atom% (15)…
A simple novel configuration for in-vial microporous membrane liquid–liquid extraction
2009
Abstract A novel arrangement for microporous membrane liquid–liquid extraction from the aqueous donor phase to the organic acceptor phase within a micro-vial, which is compatible with the chromatograph autosampler is presented. The device consisted of a stoppered glass micro-vial containing the organic solvent where the septum of the screw stopper was replaced by a sized piece of membrane which is hermetically assembled to the volumetric flask containing the aqueous donor solution. The placement of the membrane in alternative contact with the solutions was achieved by orbital agitation. As a preliminary study, 2-ethylhexyl 4-(dimethylamino)benzoate has been determined (limit of quantificati…
Determination of hydroxylated benzophenone UV filters in sea water samples by dispersive liquid-liquid microextraction followed by gas chromatography…
2010
A new analytical method for the determination of four hydroxylated benzophenone UV filters (i.e. 2-hydroxy-4-methoxybenzophenone (HMB), 2,4-dihydroxybenzophenone (DHB), 2,2'-dihydroxy-4-methoxybenzophenone (DHMB) and 2,3,4-trihydroxybenzophenone (THB)) in sea water samples is presented. The method is based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS) determination. The variables involved in the DLLME process were studied. Under optimized conditions, 1000 microL of acetone (disperser solvent) containing 60 microL of chloroform (extraction solvent) were injected into 5 mL of aqueous sample adjusted to pH 4 and containing 10% NaCl…
Comparison of different methods for extraction of polycyclic aromatic hydrocarbons (PAHs) from Sicilian (Italy) coastal area sediments.
2012
This paper describes a work aimed at improving the conditions of an extraction method, coupling GC-MS determination, for the analysis without cleanup phase, of polycyclic aromatic hydrocarbons (PAHs) from sediment samples. The automatic Soxhlet extraction in warm mode (using Extraction System B-811 Standard, Buchi) has demonstrated advantages for automation, reduced extraction time, and lower solvent use than for conventional Soxhlet extraction. Under these conditions, the recoveries are very good as they resulted greater than 85 % and, in most of the cases, near 100 %. The repeatability is also satisfactory (relative standard deviation less than 15 %). The detection limits are also accepta…
Analysis of currently used pesticides in fine airborne particulate matter (PM 2.5) by pressurized liquid extraction and liquid chromatography-tandem …
2008
During and after the application of currently used pesticides (CUPs) a significant fraction of applied pesticides can be lost to the air. A confirmatory and rapid procedure has been developed for the determination of four fungicides (carbendazim, thiabendazol, imazalil and bitertanol), three insecticides (imidacloprid, methidathion and pyriproxyfen), one helicide (methiocarb) and one acaricide (hexythiazox) in fine airborne particulate matter (PM 2.5) at trace level. The proposed method includes extraction of PM 2.5-bound pesticides by pressurized liquid extraction (PLE) followed by a direct injection into LC-MS/MS. The main parameters affecting the performance of the electrospray ionizatio…