Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “selective extraction”

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.

At least 631 records · Page 35Linked to original sources
PubMed2000

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles.

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.

Animals↗
PubMed1996

Purification of Mycoplasma gallisepticum membrane proteins p52, p67 (pMGA), and p77 by high-performance liquid chromatography.

The plasma membrane of the avian pathogen Mycoplasma gallisepticum contains about 200 polypeptides including the major lipoprotein pMGA. We have developed a simple and efficient procedure for the purification of three membrane proteins of this wall-less bacterium. Proteins were selectively extracted from isolated plasma membranes with the mild zwitterionic detergent (N-dodecyl-N,N-dimethylammonio) undecanoate (DDMAU) and subjected to size-exclusion chromatography (FPLC) in the presence of the same detergent. Two of the thus separated protein fractions were subjected to a third step involving an anion-exchange chromatography (HPLC), also in the presence of DDMAU, which led to the purification to homogeneity of p67, the major acyl protein of M. gallisepticum plasma membrane (yield, 40%; purification factor, 11), p52 (yield, 38%; purification factor, 20), and p77 (yield, approximately 45%; purification factor, 500). Analyses performed by Western blotting and crossed immunoelectrophoresis showed that the three purified proteins are distinct antigens. Furthermore, N-terminal sequencing confirmed that p67 is pMGA. The method described in this paper is simple, efficient, and nondenaturing; it provides pure proteins, at the milligram level for p52 and p67, and should prove easy to being scaled-up if necessary.

Antigens, Surface↗
PubMed2002

Purification and characterization of the major lipoprotein (P28) of Spiroplasma apis.

The plasma membrane of Spiroplasma apis contains a 28-kDa major protein (P28), like other spiroplasmas which also possess a main 26- to 28-kDa membrane polypeptide, called spiralin. In the work described here, we have developed a simple and efficient method for the purification of P28 of this mollicute, a wall-less eubacteria. Proteins were first selectively extracted from the isolated membrane with the mild detergents (i) sodium N-lauroylsarcosinate (Sarkosyl) and (ii) 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (Chaps) and subjected to size-exclusion HPLC in the presence of Chaps. The P28-enriched fraction was thereafter subjected to the second chromatographic step involving cation exchange HPLC in the presence of the same detergent. P28 was purified at the milligram level (yield, 40%). Metabolite labeling with [14C]palmitic acid and chemical analysis of P28 indicated that it is covalently modified by two O-ester-bound fatty acids and one amide-linked chain and contains a S-glycerylcysteine at the N-terminus. By charge-shift electrophoresis, Triton X-114 phase separation, and growth inhibition tests it was shown that P28 is a typical amphiphilic protein exposed, at least partly, at the cell surface. Together, our data provided evidence that P28 is a "classical" lipoprotein (i.e., triacylated) like the members of the spiralin family.

Bacterial Outer Membrane Proteins↗
PubMed1978

A very sensitive gas chromatographic method for the evaluation of styrene oxidase and styrene oxide hydratase activities.

Styrene is a compound widely used in the manufacture of polystyrenic plastics and it has recently been shown to exert mutagenic effects after metabolic activation into styrene oxide by the microsomal mixed function oxidases; this oxide is further converted into inactive styrene glycol. In order to investigate the relative importance of activation and desactivation processes of styrene, we developed a gas chromatographic method which enables us to simultaneously measure styrene oxide and styrene glycol formed after incubation of styrene with microsomal preparations from different tissues. After selective extraction of the two compounds from the incubation mixture, they are derivatized with pentafluorobenzoyl chloride and measured by gas chromatography using an electron capture detector. The high sensitivity of the method, which allows 0.01 ng of both compounds to be measured, as well as its selectiveity, has permitted us to adequately evaluate the kinetic parameters of styrene oxidase and styrene oxide hydratase activities, as well as their modifications under the influence of various pretreatments of the animals.

Animals↗
PubMed1995

An extrachromosomal fragment of telomeric DNA in wheat.

A procedure developed originally for selective extraction of viral (extrachromosomal) DNA from virus-infected mammalian cells was applied to cell nuclei isolated from uninfected wheat embryos. The resulting nuclear extrachromosomal DNA (exDNA) was enriched for telomere-type sequences by isopycnic centrifugation and inserted into the Sma I site of pUC119. A cloned DNA fragment (241 bp) was found to consist primarily of tandemly repeated heptamere units of the same sequence (5'-CCCTAAA-3') that is known to predominate in telomeric DNA of Arabidopsis thaliana. Hybridization experiments indicate that extrachromosomal telomeric repeats are abundant in resting embryos and disappear rapidly during germination.

Arabidopsis↗
PubMed1987

Binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules.

The interaction of glyceraldehyde 3-phosphate dehydrogenase with microtubules has been studied by measurement of the amount of enzyme which co-assembles with in vitro reconstituted microtubules. The binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules is a saturable process; the maximum binding capacity is about 0.1 mole of enzyme bound per mole of assembled tubulin. Half saturation of microtubule binding sites is obtained at a concentration of glyceraldehyde 3-phosphate dehydrogenase of about 0.5 microM. Glyceraldehyde 3-phosphate dehydrogenase (between 0.1 and 2 microM) induces a concentration-dependent increase a) in the turbidity of the microtubule suspension without alteration of the net amount of polymer formed and b) in the amount of microtubule protein polymers after cold microtubule disassembly. There is a linear relationship between the intensity of the glyceraldehyde 3-phosphate dehydrogenase-induced effects and the amount of microtubule-bound enzyme. The specificity of the association of glyceraldehyde 3-phosphate dehydrogenase to microtubules has been documented by copolymerization experiments. Assembly-disassembly cycles of purified microtubules in the presence of a crude liver soluble fraction results in the selective extraction of a protein with an apparent molecular weight of 35,000 identified as the monomer of glyceraldehyde 3-phosphate dehydrogenase by peptide mapping and immunoblotting. In conclusion, microtubules possess a limited number of binding sites for glyceraldehyde 3-phosphate dehydrogenase. The binding of the glycolytic enzyme to microtubules shows a considerable specificity and is associated with alterations of assembly and disassembly characteristics of microtubules.

Animals↗
PubMed1976

Trace elements analysis in biological samples by radioisotopic x-ray fluorescence.

The X-ray fluorescence technique, induced by radioisotopic sources, provides a very simple method for the simultaneous analysis of trace elements in biological samples. For blood, serum, platelets, etc., samples of about 0.1 ml were deposited on filter paper disks, dried, and analyzed. In such a way the "thin specimen" approximation is realized, resulting in the following advantages: The X-ray intensity of a given element is a liner function of mass per unit area over several orders of magnitude. Interelement effects became negligible. The ratio of fluorescent X-rays to scattered radiation is increased. The sensitivity of the technique for elements with atomic number ranging from about 20-92 varies from some units to some tens of parts per million by weight in 100 s measuring time, by using a gas proportional counter, and from about some tenths to some parts per million by using an X-ray semiconductor detector, in a measuring time of 10(3)-10(4)s. In such a way and with the described features, the Cl, K, Ca, Fe, Cu, Zn, Br content of several speciments of blood and serum was determined. Measurements were further carried out in order to labelling blood components with stable tracers and to detect their concentration by means of the X-ray fluorescence technique. The life span of platelets was, for example, measured after labelling platelets with stable Selenocystine. The sensitivity of the XRF technique can further be enhanced by about three orders of magnitude by using a pre-enrichment step with ion-exchange resins and liquid volumes not lower than 500 ml. Urine analyses have been carried in such a way, and copper in about 20 ml serum after selective extraction.

Blood Platelets↗
PubMed1987

Characterization of the major iron-regulated protein of Neisseria gonorrhoeae and Neisseria meningitidis.

The major iron-regulated protein (MIRP) was purified, from both Neisseria gonorrhoeae and N. meningitidis by selective extraction with cetyltrimethylammonium bromide followed by ion-exchange and moleculair-seive chromatography. Solutions of the purified proteins had a characteristic pink color. The overall amino acid composition of these proteins was similar, although differences were noted in the number of serine, threonine, and lysine residues. Nevertheless, the N-terminal amino acid sequence was identical through 47 residues for both the meningococcal and gonococcal MIRP. Plasma emission spectrophotometry revealed that the meningococcal 37K protein contained ca. 1 mole Fe/mole protein.

Amino Acid Sequence↗
PubMedDate not supplied

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine.

Groups of CBA mice were administered [35S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1(0) histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HCl) contained [35S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.

Age Factors↗
PubMed1986

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster.

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, "drosopterins," and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.

Amino Acids↗
PubMed1986

Chromogranin A, B and C immunoreactivities of mammalian endocrine cells. Distribution, distinction from costored hormones/prohormones and relationship with the argyrophil component of secretory granules.

Antibodies specific for chromogranin A, B or C have been used to detect immunohistochemically these three anionic proteins. Pancreatic A, B and PP cells, gut argentaffin EC, argyrophil ECL and gastrin G cells, thyroid C cells, parathyroid cells, adrenal medullary cells, pituitary TSH, FSH and LH cells as well as some axons of visceral nerves have been found to react with chromogranin A antibodies. Pancreatic A, gut EC and G, adrenal medullary and pituitary cells as well as some gut nerve fibers showed chromogranin B immunoreactivity. Chromogranin C immunoreactivity has been detected in pancreatic A, pyloric D1, intestinal L, thyroid C, adrenal medullary and pituitary cells, as well as in some gut neurons and nerve fibers. No crossreactivity has been found in immunohistochemical tests between chromogranins A, B or C and costored monoamines or peptide hormones/prohormones, from which chromogranins can be separated by selective extraction during fixation. On both morphological and chemical grounds a relationship seems to exist between chromogranin A and Grimelius' argyrophilia. Sialooligosaccharide chains of chromogranin A and, possibly, chromogranins' phosphoserine/phosphothreonine groups, seem to interact with guanidyl, amino, and/or imidazole groups of non-chromogranin components to form silver complexing sites accounting for granules' argyrophilia, which can be removed or blocked without affecting chromogranin immunoreactivities. The abundant anionic groups of the three proteins should contribute substantially to granules' basophilia, the partly "masked" pattern of which supports the existence of a close interaction of such groups with other components of secretory granules, including monoamines and peptide hormones or prohormones. Chromogranins could play a rôle in hormone postranslational biosynthesis and intragranular packaging.

Adrenal Medulla↗
PubMed1975

Assay of the major (4-hydroxylated) metabolites of diphenylhydantoin in human urine.

A modified gas chromatographic procedure for the determination of unconjugated and conjugated 4-hydroxydiphenylhydantoin (4-OH-DPH) in urine has been developed. Unconjugated 4-OH-DPH is determined after selective extraction with toluene-ether (1:1). For the assay of conjugated 4-OH-DPH, the urine is pre-extracted with isoamylalcohol before acid hydrolysis to avoid interference by the dihydrodiol metabolite of DPH. The sensitivity of the method is 0.1 mug per ml. The method has been used to determine the urinary metabolites in two adult volunteers, during steady state plasma concentrations of DPH and in the elimination phase.

Adult↗
PubMed1981

Studies on the resolution of cytochrome oxidase.

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I-IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V-VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of heme a3. It is suggested that the fraction of subunits I-IV,VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.

Acetates↗
PubMed1995

Preparation and biodistribution of yttrium-90 Lipiodol in rats following hepatic arterial injection.

In this study, we labelled Lipiodol with yttrium-90 and analysed the biodistribution in rats after intrahepatic arterial injection. An RP-18 column (E. Merck) was used to separate 90Y from strontium-90. 90Y was retained on the column, which had been pretreated with yttrium-selective extraction reagent, di(2-ethylhexyl) phosphate, while 90Sr was washed out. A hexadentate nitrogen-donor chelating ligand N,N,N',N'-tetrakis(2-benzymidazolylmethyl)-1,2-ethanediamine (EDTB) was synthesized by condensation of 1,2-benzenediamine and ethylene diamine tetra-acetic acid (EDTA). Lipiodol was covalently conjugated with EDTB. The final product was obtained by eluting the retained 90Y from the RP-18 column with EDTB-Lipiodol. Sixteen male rats (Sprague-Dawley) were sacrificed at 1 h, 24 h, 48 h and 72 h (four rats at each time) after injection of approximately 0.1 mCi 90Y-Lipiodol via the hepatic artery. Samples of liver, spleen, muscle, lung, kidney, bone, whole blood and testis were obtained and counted to calculate the tissue concentrations. In addition, labelling efficiency and in vitro stability were determined by ITLC methods. We found that at 1 h after intrahepatic injection, most of the radiotracer was retained in the liver, but it was eliminated gradually over a few days. The radioactivity level in the lung was fair at 1 h and remained at roughly the same level throughout the study. Radioactivity in the kidney and spleen reached a relatively high level at 24 h, but declined rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

A method for distinction between RNA and DNA in aldehyde and osmiumtetroxide-fixed electron microscopic autoradiographs.

A simple method for distinction between RNA- and DNA-containing structures in aldehyde- and osmiumtextroxide-fixed electron microscopic autoradiographs (or ordinary thin sections) is described: the developer and the acetic acid used for processing autoradiographs extract selectively uranium acetate from DNA containing-structures which, after staining with lead citrate, leads to a characteristically 'bleached' appearance of the DNA.

Aldehydes↗
PubMed1991

Effect of alpha-tocopherol and di-butyl-hydroxytoluene (BHT) on UV-A-induced photobinding of 8-methoxypsoralen to Wistar rat epidermal biomacromolecules in vivo.

The possible formation of singlet oxygen via photoexcited psoralens has been associated with the occurrence of, amongst others, erythema. Therefore it has been suggested to combine PUVA with the topical or systemic administration of antioxidants. However, the effect of these antioxidants on the photobinding of psoralens to DNA, which is held responsible for the anti-proliferative effect, should be taken into account. In the present study the effect of two phenolic antioxidants, alpha-tocopherol (AT) and butylated hydroxytoluene (BHT), on the in vivo photobinding of 8-methoxypsoralen (8-MOP) to not only epidermal DNA, but also proteins and lipids was determined. After topical application of an ethanolic antioxidant solution onto the shaven skin of Wistar rats, labeled 8-MOP was applied using the same solvent. After this the rats were exposed to UV-A. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP to each of these biomacromolecules was determined. Both AT and BHT caused a decrease in the photobinding of 8-MOP to epidermal DNA and proteins. To investigate the underlying mechanism of this protection, the effect of AT was compared with that of AT-acetate. It also proved helpful to study the effects of the antioxidants on the photobinding of another photosensitizer, namely chlorpromazine. From these experiments it was concluded that AT and BHT affect 8-MOP photobinding by quenching reactive 8-MOP intermediates, involving the phenolic hydroxyl group of the antioxidants. BHT offered protection against lipid binding of 8-MOP but AT, especially at high concentrations, enhanced the UV-A-induced binding of 8-MOP to lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

[On the detection of clomethiazole in urine (author's transl)].

It may become necessary to prove that clomethiazole (Distraneurin) has been taken when supervising addicts, with traffic accidents under the influence of drugs and when the cause of death is unclear. Since the compound is almost completely metabolized in the body, a method has been developed to detect its main metabolite, 4-methyl-5-thiazole acetic acid in the urine. The acid can be precisely identified and determined by a combination of selective extractions, high-voltage electrophoresis and UV measurements.

Chlormethiazole↗
PubMed1982

Differential uptake of cholesterol and plant sterols by rat erythrocytes in vitro.

The in vitro uptake of radioactively labeled cholesterol and the plant sterol beta-sitosterol has been examined in rat erythrocytes. From mixed micellar solutions containing egg yolk phospholipid and sodium taurocholate, the erythrocytes showed a nonlinear uptake of the two sterols. The uptake leveled off after about 45 min with the attainment of a 1:1 total sterol-to-phospholipid ratio within the cell membrane, as determined on a mass basis. From solutions containing egg yolk phospholipid, or purified egg yolk phosphatidylcholine, a preference for cholesterol over the plant sterol was observed, increasing with time from a cholesterol/beta-sitosterol uptake ratio of unity (the media ratio) to a maximum of 2 after a 60-min incubation. Correction of the data for nonspecifically bound sterol increased the ratio to a maximum of 5 at the 30-min time point. The increase in the cholesterol/beta-sitosterol uptake ratio with time, following an initial nonspecific association, showed that penetration of the plasma membrane by the sterol was required for the selectivity to be expressed. The presence of lysophosphatidylcholine or bovine serum albumin did not exert any noticeable influence over the extent or selectivity of absorption. Replacement of the egg yolk phospholipid with synthetic dipalmitoyl-phosphatidylcholine led to a loss of the sterol selectivity. No evidence was found to support a selective extraction of sterol from the erythrocyte membrane to account for the observed effects, nor was there any sign of a mass accumulation of phospholipid during the incubation. It is suggested that the media phospholipid influences the membrane permeability toward cholesterol and beta-sitosterol.

Animals↗