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PubMed1976

Non-lytic, non-ionic detergent extraction of plasma membrane constituents from normal and transformed fibroblasts.

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cell employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, 51Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodination followed by detergent extraction permits demonstration of a 100,000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.

Cell Fractionation↗
PubMed2003

The use of extraction chromatography resins to concentrate actinides and strontium from soil for radiochromatographic analyses.

An analytical technique utilizing selective extractant resins to concentrate strontium and actinides from soil followed by separation with radiochromatography was evaluated. The technique was tested using uncontaminated soil samples spiked with a radionuclide tracer solution that were either microwave-aided acid digested or leached with a strong acid. Extraction of the strontium and actinides from the acidified solution was accomplished using a serial arrangement of Sr-Resin and TRU-Resin columns. The combined eluate solutions from the extraction resins were treated with HNO(3) and H(2)O(2) to oxidize residual extractant and eluates prior to separation and analysis of the radionuclides by radiochromatography. Chromatograms obtained with larger soil mass loadings resulted in either incomplete peak resolution of the tracers or had highly variable peak elution times, indicative of an ionic interfering constituent(s). Better separations (e.g., chromatograms that resolved all radioactive constituents) were obtained when the sample mass loading was decreased, but with a concurrent decreased sensitivity for the radionuclides. Elemental analyses of the soil were conducted to provide data on the ionic constituents in unprocessed soil and post-processed soil samples. These results identified aluminum as an interfering contributor to the poor performance exhibited by the radiochromatographic separations.

Actinoid Series Elements↗
PubMed1982

Gallamine blue staining of DNA in mammalian tissue sections: analyses of in situ absorption spectra.

This paper reports on the use of gallamine blue (GB), a dye of the oxazine group, as a specific stain for DNA in animal tissue nuclei. The dye can be used as 1% aqueous solution in boiling distilled water at ph 1.0 to 1.5. Not only this, GB dye-reagent can also be prepared after dispersing the dye with concentrated sulphuric acid and then dissolving the friable mass in 1% cobalt chloride and then used to stain nuclei at very low pH. Although the dye does not contain any primary amino group in its molecules, it can be used as aqueous solution or as dye-reagent to stain DNA-aldehyde molecules in tissue sections which are hydrolysed in 6N HCl at 28 degrees C or at 40 degrees C for 15 and 5 min, respectively. Following staining of the DNA-aldehyde molecules, the preparations cannot be treated with SO2 water, since this treatment brings about complete leaching of the dye from the nuclei. It has, therefore, been concluded that GB staining of DNA-aldehyde molecules is due to a modified Feulgen reaction in which tertiary amino group may be involved. Moreover, GB in an aqueous solution or as a dye-reagent can be used to stain DNA-phosphate groups in tissue sections from which RNA has been extracted selectively with cold concentrated phosphoric acid. Sections from which RNA has been extracted and then hydrolysed in 6N HCl at 28 degrees C or at 40 degrees C for 15 and 5 min, respectively, can also be stained with this dye. The absorption spectra of nuclei stained following the various procedures have been presented. The paper contains a discussion on the implications of all these findings.

Animals↗
PubMed2001

Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.

Chlorophyll↗
PubMed1987

Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin.

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 micron in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In contrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower Mr proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.

Animals↗
PubMed2006

Flow injection on-line solid phase extraction coupled with inductively coupled plasma mass spectrometry for determination of (ultra)trace rare earth elements in environmental materials using maleic acid grafted polytetrafluoroethylene fibers as sorbent.

A new sorbent, maleic acid grafted polytetrafluoroethylene fiber (MA-PTFE), was prepared and evaluated for on-line solid-phase extraction coupled with inductively coupled plasma mass spectrometry (ICP-MS) for fast, selective, and sensitive determination of (ultra)trace rare earth elements (REEs) in environmental samples. The REEs in aqueous samples at pH = 3.0 were selectively extracted onto a microcolumn packed with the MA-PTFE fiber, and the adsorbed REEs were subsequently eluted on-line with 0.9 mol l(-1) HNO3 for ICP-MS determination. The new sorbent extraction system allows effective preconcentration and separation of the REEs from the major matrix constituents of alkali and alkali earth elements, particularly their separation from barium that produces considerable isobaric interferences of 134Ba16O1H+, 135Ba16O+, 136Ba16O1H+, and 137Ba16O+ on 151Eu+ and 153Eu+. With the use of a sample loading flow rate of 7.4 ml min(-1) for 120 s preconcentration, enhancement factors of 69-97 and detection limits (3s) of 1-20 pg l(-1) were achieved at a sample throughput of 22 samples h(-1). The precision (RSD) for 16 replicate determinations of 50 ng l(-1) of REEs was 0.5-1.1%. The developed method was successfully applied to the determination of (ultra)trace REEs in sediment, soil, and seawater samples.

Journal Article↗
PubMed1996

Reconstruction of alveolar bone defects after extraction of mandibular third molars: a pilot study.

INTRODUCTION: A documented complication of mandibular third molar extraction is the development of bony defects on the distal aspect of the adjacent second molar. The primary study purpose was to determine the efficacy of grafting third molar extraction sites with demineralized bone powder to prevent the formation of periodontal defects. MATERIAL/METHODS: With a randomized clinical trial study design and a third molar extraction surgical model, we enrolled a study sample composed of patients who required extraction of bilateral third molars. Demineralized bone powder was placed in one randomly selected extraction site per patient. The remaining extraction site served as a control. Patients served as their own controls. To assess periodontal healing, we measured plaque and gingival indexes and periodontal attachment loss on the distobuccal aspect of the adjacent second molar preoperatively and 6 months postoperatively. RESULTS: Of 14 patients enrolled, 7 patients with a mean age of 21.7 +/- 3.7 years completed the study protocol. No statistically significant differences were noted between patients who did and did not complete the study protocol (all p values > or = 0.42). There were noted statistically significant differences for the various anatomic, operative, or periodontal measures between the control and treatment teeth preoperatively (all p values > or = 0.46). Six months postoperatively, there were no statistically significant changes in the plaque or gingival indexes for the control or experimental sites. At the control sites, there was a nonstatistically significant decrease in mean attachment loss from 3.1 +/- 1.3 mm preoperatively to 1.4 +/- 1.6 mm 6 months postoperatively (p = 0.06). At experimental sites, there was a statistically significant decrease in mean attachment loss from 3.3 +/- 1.4 mm preoperatively to 0.6 +/- 0.8 mm 6 months postoperatively (p = 0.02) CONCLUSIONS: The study results suggest that demineralized bone powder may decrease attachment loss on the distal aspect of the second molar after extraction of the adjacent third molar. Additional studies are indicated to confirm the results of this pilot study. We recommend that future studies limit their study samples to patients at high risk for developing periodontal defects after third molar extraction.

Adult↗
PubMed1982

Specific recognition of pyrophosphate in nucleotides.

The lipophilic diammonium cation 1 was demonstrated to be a reagent to discriminate the number of the phosphate in nucleotides. Thus the highly hydrophilic nucleotides were selectively extracted into an organic phase by the formation of ion-pairs and selectively transported through a membrane by a carrier mechanism. The compound was also shown to be an efficient phase transfer reagent which activated the phosphate anion of AMP to give a facile synthesis of pyrophosphate linkages, ADP and ATP, without any protection of the sugar moiety.

Adenine Nucleotides↗
PubMed1996

Increased rotational mobility and extractability of band 3 from protein 4.2-deficient erythrocyte membranes: evidence of a role for protein 4.2 in strengthening the band 3-cytoskeleton linkage.

Band 3 (anion-exchange protein 1-[AE1]) is the major integral membrane protein of human erythrocytes and links the membrane to the underlying cytoskeleton via high-affinity binding to ankyrin. It is unclear whether other cytoskeletal proteins participate in strengthening the ankyrin-band 3 linkage, but a putative role for protein 4.2 (P4.2) has been proposed based on the increased osmotic fragility and spherocytic morphology of P4.2-deficient red blood cells (RBCs). The present study was designed to investigate the hypothesis that P4.2 has a direct role in strengthening the band 3-cytoskeleton linkage in human RBCs, by measuring independent features of this interaction in normal and P4.2-deficient RBCs. The features examined were the rotational mobility of band 3 assayed by time-resolved phosphorescence emission anisotropy (TPA), and the extractability of band 3 by octyl-beta-glucoside, the latter being a nonionic detergent that selectively extracts only band 3 that is not anchored to the cytoskeleton. We find that the amplitude of the most rapidly rotating population of band 3 (correlation time, approximately 30 to 60 microseconds) is increased 81% and 67% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts. The amplitude of the intermediate speed rotating population of band 3 (correlation time, approximately 200 to 500 microseconds) is increased 23% and 8% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts, at the expense of the slowly rotating component (correlation time, approximately 2,000 to 3,000 microseconds, amplitude decreased 43% and 39% in P4.2NIPPON and band 3MONTEFIORE, respectively) and immobile component (immobile on this experimental time scale; amplitude decreased 26% and 10% in P4.2NIPPON and band 3MONTEFIORE, respectively) of band 3. These results demonstrate that P4.2 deficiency partially removes band 3 rotational constraints, ie, it increases band 3 rotational mobility. The nonionic detergent octyl-beta-glucoside, which does not disturb band 3-cytoskeleton associations, ie, it extracts only band 3 that is not attached to the cytoskeleton, extracted 30% and 61% more band 3 from P4.2NIPPON and band 3MONTEFIORE ghost membranes, respectively, compared with control ghosts. The octyl-beta-glucoside ghost extracts from both P4.2-deficient phenotypes were enriched in band 3 oligomeric species (tetramers, higher-order oligomers, and aggregates) compared with controls. Since band 3 oligomers selectively associate with the cytoskeleton, these results are consistent with a weakened band 3-cytoskeleton linkage in P4.2-deficient RBC membranes. P4.2 deficiency does not affect band 3 anion transport activity, since uptake of radiolabeled sulfate was similar for control and P4.2-deficient RBCs. Thus, we propose that P4.2 directly participates in strengthening the band 3-cytoskeleton linkage.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2005

Development and application of a sol-gel immunosorbent-based method for the determination of isoproturon in surface water.

An immunosorbent was fabricated by encapsulation of monoclonal anti-isoproturon antibodies in sol-gel matrix. The immunosorbent-based loading, rinsing and eluting processes were optimized. Based on these optimizations, the sol-gel immunosorbent (SG-IS) selectively extracted isoproturon from an artificial mixture of 68 pesticides. In addition to this high selectivity, the SG-IS proved to be reusable. The SG-IS was combined with liquid chromatography-tandem mass spectrometry (LC-MS-MS) to determine isoproturon in surface water, and the linear range was up to 2.2 microg/l with correlation coefficient higher than 0.99 and relative standard deviation (RSD) lower than 5% (n=8). The limit of quantitation (LOQ) for 25-ml surface water sample was 5 ng/l.

Calibration↗
PubMed1982

Assay of bromhexine in human plasma by capillary gas--liquid chromatography with nitrogen-selective detection and selected ion monitoring.

A specific, sensitive method for the determination of bromhexine in human plasma is described. It comprises a selective extraction procedure and a specific determination with capillary gas--liquid chromatography and nitrogen-selective flame ionization detection. The detection limit of the assay is about 0.5 ng/ml. The specificity of the assay was checked by gas chromatography--mass spectrometry. The method is applied to the pharmacokinetics of bromhexine in humans.

Adult↗
PubMed1983

Purification and characterization of two forms of DNA polymerase alpha from HeLa cell nuclei.

Isolated nuclei contained two active forms of DNA polymerase alpha (form I and form II). Form II was extracted from nuclei by KCl at concentrations lower than 0.18 M. Above 0.18 M selective extraction of form I was observed. The purified two active forms differed in chromatographic and electrophoretic behaviors, in their salt requirement for optimal activity, and in preference of template-primers, although both forms exhibited properties characteristic of DNA polymerase alpha such as sensitivity of N-ethylmaleimide, 1-beta-D-arabino-furanosylcytosine triphosphate, and aphidicolin. Marked difference between the two forms was preference of template-primer that form I was more active with poly(dT).(rA)10 than poly(dA).(dT)12 whereas form II exhibited higher activity with poly(dA).(dT)12 than poly(dT).(rA)10. Possible roles of two forms of DNA polymerase alpha in the processes of DNA replication will be discussed.

Cell Nucleus↗
PubMed1974

Preparation of cells permeable to macromolecules by treatment with toluene: studies of transfer ribonucleic acid nucleotidyltransferase.

Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl(2), or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent K(m) values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5'-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.

Adenosine↗
PubMed1985

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography.

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.

Animals↗
PubMed1989

[Solvent extraction and high performance liquid chromatography with electrochemical detection for determination of plasma catecholamines].

This paper describes a technique for selectively extracting plasma catecholamines prior to quantification by HPLC-EC. The extraction system was a two-stage process. The first stage involve the complex formation between diphenylbrorate and catechol (diol) groups in alkaline medium. The second stage was a liquid-liquid extraction. The complex combined with tetraoctylammonium bromide to form an ion-pair formation into organic solvent. The catecholamines in turn was extracted with acid. This technique provided a very specific extraction procedure which resulted in chromatograms with few interfering compounds and gave absolute recoveries (100-103%) of norepinephrine, epinephrine and dopamine. Meanwhile, the plasma catecholamines were concentrated and the detective sensitivity was increased. A good linear relationship was found between the concentrations and ratio of peak heights of the catecholamines from 0.125-2 ng. The correlation coefficients ranged from 0.998-0.999. The coefficients of variation of the intra- and inter-assay were within 3 and 6% respectively. The results show that the procedure is very simple and fast. The method is valuable not only for clinical diagnosis but also for laboratory research.

Catecholamines↗
PubMed2007

Spontaneous reconstitution of discoidal HDL from sphingomyelin-containing model membranes by apolipoprotein A-I.

Nascent HDL is known to be formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ABCA1, but the molecular mechanism by which nascent HDL forms is less well understood. Here, we studied how reconstituted high density lipoprotein (rHDL) forms spontaneously on the interaction of apoA-I with model membranes. The formation of rHDL from pure phosphatidylcholine (PC) large unilamellar vesicles (LUVs) proceeded very slowly at 37.0 degrees C, but sphingomyelin (SM) -rich PC/SM LUVs, which are in a gel/liquid-disordered phase (L(d) phase) at this temperature, were rapidly microsolubilized to form rHDL by apoA-I. The addition of cholesterol decreased the rate at which rHDL formed and induced the selective extraction of lipids by apoA-I, which preferably extracted lipids of L(d) phase rather than lipids of liquid-ordered phase. In addition, apoA-I extracted lipids from the outer and inner leaflets of LUVs simultaneously. These results suggest that the heterogeneous interface of the mixed membranes facilitates the insertion of apoA-I and induces L(d) phase-selective but leaflet-nonselective lipid extraction to form rHDL; they are compatible with recent cell works on apoA-I-dependent HDL generation.

Apolipoprotein A-I↗
PubMed2006

Effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley (Hordeum vulgare L.).

Four kinds of solvent extracts from three Chinese barley varieties (Ken-3, KA4B, and Gan-3) were used to examine the effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley through free radical scavenging activity, reducing power and metal chelating activity, and individual and total phenolic contents. Results showed that extraction solvent mixtures had significant impacts on antioxidant activity estimation, as well as different extraction capacity and selectivity for free phenolic compounds in barley. The highest DPPH* and ABTS*+ scavenging activities and reducing power were found in 80% acetone extracts, whereas the strongest *OH scavenging activity, O2*- scavenging activity, and metal chelating activity were found in 80% ethanol, 80% methanol, and water extracts, respectively. Additionally, 80% acetone showed the highest extraction capacity for (+)-catechin and ferulic, caffeic, vanillic, and p-coumaric acids, 80% methanol for (-)-epicatechin and syringic acid, and water for protocatechuic and gallic acids. Furthermore, correlations analysis revealed that TPC, reducing power, DPPH* and ABTS*+ scavenging activities were well positively correlated with each other (p < 0.01). Thus, for routine screening of barley varieties with higher antioxidant activity, 80% acetone was recommended to extract free phenolic compounds from barley. DPPH* scavenging activity and ABTS*+ scavenging activity or reducing power could be used to assess barley antioxidant activity.

Acetone↗