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Anion Exchange (anion + exchange)
Terms modified by Anion Exchange Selected AbstractsExtensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patientsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 1 2009Linda M. Benson Abstract Allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma are prevalent airway diseases that can have a substantial impact on a patient's quality of life. MS analyses of biological fluids can effectively screen for proteins associated with disease processes, however, initial detection of diagnostic proteins is difficult due to protein complexity and dynamic range. To enhance the detection of lower abundance proteins, intact nasal lavage fluid (NLF) proteins from nonpolypoid AR and from asthmatic CRS patients were extensively fractionated prior to LC/MS/MS analysis. Pooled NLF samples were processed to remove low molecular weight molecules and high abundance plasma proteins. Anion exchange (AX) chromatography followed by RP-LC further separated the remaining intact NLF proteins. The resulting fractions were digested with trypsin and the peptides analyzed by LC/MS/MS. Spectra were searched with MASCOT, SEQUEST, and X!Tandem to obtain peptide identifications and subsequently analyzed by Scaffold software to identify parent proteins with at least 99% confidence. The 197 identified proteins are compared to those previously cited in the literature and the workflow evaluated to determine the usefulness for the detection of lower abundance proteins. This is the first extensive list of NLF proteins generated from CRS patients with coexisting asthma. [source] Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumicanTHE PROSTATE, Issue 3 2004John W. Holland Abstract BACKGROUND Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65,95 kDa. The KS moiety was susceptible to digestion with keratanase II and peptide N -glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation. © 2004 Wiley-Liss, Inc. [source] Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheatARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Charles Darkoh Abstract Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu2+ sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5,U/mg of total protein for gluten in a SDS gluten assay (1,U of enzyme activity was defined as the decrease in gel height in millimeters in 1,h) and exhibited a high-affinity Km of 65,µM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20° and 35°C. The enzyme was identified to be a prolyl endoprotease. © 2010 Wiley Periodicals, Inc. [source] Interactions of Cationic Palladium(II)- and Platinum(II)-,3 -Allyl Complexes with Fluoride: Is Asymmetric Allylic Fluorination a Viable Reaction?EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 7 2006Lukas Hintermann Abstract The complex cations [M(,3 -R2All)(PPFPz{3- tBu})]+ (M = PdII, R2All = 1,3-diphenylallyl, 1,3-dicyclohexylallyl, indenyl; M = PtII, R2All = 1,3-diphenylallyl; PPFPz-{3- tBu} = 3- tert -butyl-1-{1-[2-diphenylphosphanyl-ferrocenyl]ethyl}-1H -pyrazole)have been prepared as salts with PF6, or SbF6,. They have been characterized by NMR spectroscopy in solution and by X-ray crystallography in the solid state. Their reactions with sources of nucleophilic and "naked" fluoride have been investigated by multinuclear NMR spectroscopy. The PdII complexes did not undergo any nucleophilic substitution with concomitant release of allyl fluorides. The dicyclohexylallyl fragment was released as a 1,3-diene by elimination, but with other allyl complexes nonspecific decomposition reactions predominated. The complex [Pt(,3 -1,3-Ph2C3H3)(PPFPz{3- tBu})]PF6 underwent an anion exchange with Me4NF to give [Pt(1,3-Ph2C3H3)(PPFPz{3- tBu})]F which existed as a mixture of interconverting allyl isomers in solution at ambient temperature. For the bromide salt, [Pt(,3 -1,3-Ph2C3H3)(PPFPz{3- tBu})]Br, allyl isomerization was slow at ambient temperature. Precursors of Pt0 reacted with bromo-1,3-diphenylprop-2-ene to give [Pt2(,-Br)2(,3 -1,3-Ph2All)2] and precursors of Pd0 underwent oxidative additions with bromo- and fluoro-1,3-diphenyl-2-propene to give 1,3-diphenylallyl complexes of PdII. Therefore, the nucleophilic attack of fluoride on the allyl fragment of PdII complexes is endergonic, and the high energy barrier of this step is difficult to overcome in a catalytic allylic fluorination reaction. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Synthesis and Characterization of Pyrazolyl-Functionalized Imidazolium-Based Ionic Liquids and Hemilabile (Carbene)palladium(II) Complex Catalyzed Heck ReactionEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 4 2007Ruihu Wang Abstract Neat reactions of 1-(pyrazolylmethyl)imidazole with an excess of alkyl or polyfluoroalkyl halides at 100 °C followed by subsequent metathetical reactions with LiN(SO2CF3)2 or KPF6 at 25 °C gave rise to a series of monoquaternary salts 3a,3k. These salts can be also prepared through treatment of 1-alkylimidazole with 1-(chloromethyl)pyrazole hydrochloride in the presence of base, followed by anion exchange with LiN(SO2CF3)2 or KPF6. Their phase-transition temperature, thermal stability, density and solubility in common solvents have been investigated. Most of the bis(trifluoromethanesulfon)amide salts are room-temperature ionic liquids. The effect of anions and of alkyl substituents bonded to the imidazolium cation on the physicochemical properties was examined. Using 3-butyl-1-(pyrazolylmethyl)imidazolium chloride (2d), the precursor of 3-butyl-1-(pyrazolylmethyl)imidazolium bis(trifluoromethanesulfon)amide (3d), as a reactant, a hemilabile (N-heterocyclic carbene)palladium(II) complex 4 was synthesized through a (carbene)silver(I) transfer reagent. It was characterized by single-crystal X-ray diffraction analysis. The catalytic activity and recyclability of 4 in 3d were preliminarily evaluated by consecutive Heck reactions using different substrates. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Molecular physiology of SLC4 anion exchangersEXPERIMENTAL PHYSIOLOGY, Issue 1 2006Seth L. Alper Plasmalemmal Cl,,HCO3, exchangers regulate intracellular pH and [Cl,] and cell volume. In polarized epithelial cells, they contribute also to transepithelial secretion and reabsorption of acid,base equivalents and of Cl,. Members of both the SLC4 and SLC26 mammalian gene families encode Na+ -independent Cl,,HCO3, exchangers. Human SLC4A1/AE1 mutations cause either the erythroid disorders spherocytic haemolytic anaemia or ovalocytosis, or distal renal tubular acidosis. SLC4A2/AE2 knockout mice die at weaning. Human SLC4A3/AE3 polymorphisms have been associated with seizure disorder. Although mammalian SLC4/AE polypeptides mediate only electroneutral Cl,,anion exchange, trout erythroid AE1 also promotes osmolyte transport and increased anion conductance. Mouse AE1 is required for DIDS-sensitive erythroid Cl, conductance, but definitive evidence for mediation of Cl, conductance is lacking. However, a single missense mutation allows AE1 to mediate both electrogenic SO42,,Cl, exchange or electroneutral, H+ -independent SO42,,SO42, exchange. In the Xenopus oocyte, the AE1 C-terminal cytoplasmic tail residues reported to bind carbonic anhydrase II are dispensable for Cl,,Cl, exchange, but required for Cl,,HCO3, exchange. AE2 is acutely and independently inhibited by intracellular and extracellular H+, and this regulation requires integrity of the most highly conserved sequence of the AE2 N-terminal cytoplasmic domain. Individual missense mutations within this and adjacent regions identify additional residues which acid-shift pHo sensitivity. These regions together are modelled to form contiguous surface patches on the AE2 cytoplasmic domain. In contrast, the N-terminal variant AE2c polypeptide exhibits an alkaline-shifted pHo sensitivity, as do certain transmembrane domain His mutants. AE2-mediated anion exchange is also stimulated by ammonium and by hypertonicity by a mechanism sensitive to inhibition by chelation of intracellular Ca2+ and by calmidazolium. This growing body of structure,function data, together with increased structural information, will advance mechanistic understanding of SLC4 anion exchangers. [source] Fluorescent Imaging: Surface Modification of Exfoliated Layered Gadolinium Hydroxide for the Development of Multimodal Contrast Agents for MRI and Fluorescence Imaging (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Mater. The synthesis of a new stable colloid of fluorescent LGdH layers through a newly developed surface modification method is reported by Y.-S. Yoon et al. on page 3375. This new method involves layer exfoliation, anion exchange, and PEG coating. The efficient MRI contrast-enhancement of these LGdH layers, both in vitro and in vivo, demonstrates their potential utility as a multimodal probe combining optical and MR imaging. [source] Vitronectin in clotting factor IX concentratesHAEMOPHILIA, Issue 3 2001D. Josic Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here. [source] Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger,HEPATOLOGY, Issue 2 2006Jesús M. Banales Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR),dependent Cl, efflux and subsequent biliary HCO3, secretion, possibly via Cl,/HCO3, anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl,/HCO3, exchange in secretin-stimulated HCO3, secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3, and Cl, excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl, channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl,/HCO3, exchange inhibitor 4,4,-diisothiocyanatostilbene-2,2,-disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3, and Cl,. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3, excretion but not the increased Cl, excretion, revealing a role of biliary Cl,/HCO3, exchange in secretin-induced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+ -independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl,/HCO3, exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride,bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266,275.) [source] Isolation and characterization of a protease from Pseudomonas fluorescens RO98JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2000R. Koka Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 °C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 °C, and pH 4·5 and 9·0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5·0 and 35 °C, respectively. It was heat stable with a D55 of 41 min and a D62·5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144·28, 18·73, 110·20 and 35·23 µmol were obtained for whole, ,-, ,- and ,-casein, with a Vmax of 8·26, 0·09, 0·42 and 0·70 µmol mg,1 min,1, respectively. The enzyme hydrolysed ,-casein preferentially when incubated with artificial casein micelles. [source] ,-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoformJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2003Paulo Roberto Moura Lima Abstract The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the ,-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with ,-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell,cell contact and attachment of the sarcomere to the plasma membrane. © 2003 Wiley-Liss, Inc. [source] A gemini amphiphilic phase transfer catalyst for dark singlet oxygenationJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 7-8 2008Cédric Borde Abstract A new gemini surfactant phase transfer catalyst, namely diethyl-ether-,,,-bis-(dimethyldodecylammonium molybdate) codified as 12-EO-12-Mo, was prepared by anion exchange from the analogous gemini dichloride (12-EO-12-Cl2). The physico-chemical properties of these compounds such as Krafft temperature, critical micelle concentration, surface activity and binary water-surfactant behavior were compared and the influence of the molybdate counterion was examined. Though both compounds are highly hydrophilic, the cmc of 12-EO-12-Mo (0.4,mmol L,1) is about five times lower than of its dichloride analogue (2.2,mmol L,1). Moreover, 12-EO-12-Mo exhibits an additional cubic liquid crystal phase between 53 and 64,wt%. The usefulness of 12-EO-12-Mo as an amphiphilic phase transfer catalyst for the dark singlet oxygenation was demonstrated with the peroxidation of two typical organic substrates: ,-terpinene which reacts with 1O2 according to a [4,+,2] cycloaddition and the less reactive ,-citronellol, which provides two hydroperoxides according to the ene-reaction. 12-EO-12-Mo provides a simple reaction medium with only three components for the preparative peroxidation of hydrophobic substrates by chemically generated singlet oxygen. Copyright © 2008 John Wiley & Sons, Ltd. [source] PEDOT:Poly(1-vinyl-3-ethylimidazolium) dispersions as alternative materials for optoelectronic devicesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 9 2008Cristina Pozo-Gonzalo PEDOT formulations in acetonitrile have been synthesised using a phase transfer reaction involving two steps; being a polymerization in water and then a subsequent coagulation of the corresponding PEDOT by anion exchange. The strategy followed is to synthesise organic formulations using a less acidic stabilizer and fine-tuned hydrophobic properties which are suitable for using in OLEDs as a HTL (hole transport layer), leading to an interesting starting point for OLED applications. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] [source] Enrichment and low-level determination of glyphosate, aminomethylphosphonic acid and glufosinate in drinking water after cleanup by cation exchange resinJOURNAL OF SEPARATION SCIENCE, JSS, Issue 8 2010Markus Küsters Abstract For the determination of glyphosate, aminomethylphosphonic acid and glufosinate in drinking water, different procedures of enrichment and cleanup were examined using anion exchange or SPE. In many cases interactions of, e.g. alkaline earth metal ions especially calcium could be observed during enrichment and cleanup resulting in loss of analytes. For that reason, a novel cleanup and enrichment procedure for the determination of these phosphonic acid herbicides has been developed in drinking water using cation-exchange resin. In summary, the cleanup procedure with cation-exchange resin developed in this study avoids interactions as described above and is applicable to calcium-rich drinking water samples. After derivatization with 9-fluorenylmethylchloroformate followed by LC with fluorescence detection, LOD of 12, 14 and 12,ng/L and mean recoveries from real-world drinking water samples of 98±9, 100±16 and 101±11% were obtained for glyphosate, aminomethylphosphonic acid and glufosinate, respectively. The low LODs and the high precision permit the analysis of these phosphonic acid herbicides according to the guidelines of the European Commission. [source] Monolithic poly(glycidyl methacrylate- co -divinylbenzene) capillary columns functionalized to strong anion exchangers for nucleotide and oligonucleotide separationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 16 2006Wolfgang Wieder Abstract In the present work, poly(glycidyl methacrylate- co -divinylbenzene) monoliths were synthesized and further derivatized to obtain strong anion exchange supports. Capillary monoliths (65×0.2 mm id) were prepared in situ by copolymerization of glycidyl methacrylate and divinylbenzene, employing 1-decanol and tetrahydrofuran as porogens. The free epoxy groups were derivatized in a two step synthesis to obtain quaternary ammonium functionalities. On testing the pressure stability of the synthesized monolith, a highly linear dependence between flow rate and pressure drop was obtained, indicating the high stability of the material even at high flow rates. The morphology of the copolymer was investigated by scanning electron microscopy. Mercury intrusion porosimetry showed a narrow pore size distribution, having a maximum at 439 nm. On recording a van Deemter plot the number of theoretical plates per meter was found to be 59 324. The produced strong anion exchange monoliths turned out to be highly suitable for the separation of nucleotides and oligonucleotides. [source] Heat-induced denaturation impairs digestibility of legume (Phaseolus vulgaris L and Vicia faba L) 7S and 11S globulins in the small intestine of ratJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 1 2005M Carbonaro Abstract 7S globulin from common bean (Phaseolus vulgaris L) and 11S globulin from faba bean (Vicia faba L) were isolated to over 90% purity and the digestibility of the proteins, either in native or denatured (120 °C, 20 min, 1 atm) state, was tested in the small intestine of growing rats in acute (1 h) experiments. Native globulins were well digested (92 and 95% for 7S and 11S proteins, respectively). However, after thermal denaturation, protein digestibility of 7S globulin was reduced to 88%, while that of 11S globulin to only 79%. SDS-PAGE revealed that high amounts of the intermediate proteolytic products of phaseolin (MW 22 000,27 000 Da) were present in the small intestine of rats after 1 h digestion of the denatured 7S globulin, while protein material in the high MW range (>55 000 Da) were recovered from the 11S globulin. The overall negative charge of unavailable proteins from the 7S globulin was found by anion exchange,FPLC separation to be higher than that of products from the 11S globulin. MALDI-MS analysis of proteins in the small intestine confirmed the presence of half-size phaseolin subunits (MW 23 700 Da) as breakdown products from the denatured 7S globulin, and of highly hydrophobic basic subunits (MW 20 000 Da) from the 11S globulin. Copyright © 2004 Society of Chemical Industry [source] Cell wall polysaccharides of bush butter (Dacryodes edulis (G Don) HJ Lam) fruit pulp and their evolution during ripeningJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 8 2001Crépin Ella Missang Abstract Cell wall material was isolated as alcohol-insoluble solids (AIS) from bush butter endocarp tissue at different stages of ripeness. AIS were then extracted with 0.05,M CDTA followed by increasing concentrations of KOH (0.05, 1 and 4,M respectively). The chemical extractions solubilised a total of 51.6,60.6% of AIS, the yields of CDTA extracts accounting for approximately 9.6,12.2% of AIS. The extracts as well as the residues were analysed for their sugar composition and protein and starch contents. CDTA extracted the bulk of uronic acid in AIS, but the uronic acid content (after dialysis) of these extracts showed a significant decrease as the fruits ripened (from 439 to 252,mg,g,1 between the first and the last degree of ripeness). Analysis of the CDTA extracts by anion exchange and size exclusion chromatography showed a gradual appearance of new pectic populations at low degrees of methylation and low molecular weights, indicating that CDTA-soluble pectins are demethylated and depolymerised during ripening. The dilute alkali (0.05,M KOH) extracts were essentially composed of proteins in addition to a minor quantity of pectin. The 1,M KOH and principally 4,M KOH treatments led to the extraction of hemicelluloses, mainly xyloglucan-like and mannan-like polymers. These extracts also contained substantial amounts of protein and starch. No variation related to the degree of ripeness was visible in the sugar composition of the alkali extracts. The molecular weight distribution of the hemicelluloses did not change with the degree of ripeness. The final residues accounted for 21.4,27.3% of AIS and were mostly composed of glucose (827,908,mg,g,1). All these results suggested that only CDTA-soluble pectins were involved in bush butter fruit softening. © 2001 Society of Chemical Industry [source] Changes in the pectic fraction of bush butter (Dacryodes edulis (G Don) HJ Lam) fruit pulp during ripeningJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 8 2001Crépin Ella Missang Abstract CDTA-soluble polysaccharides were extracted from cell wall material (prepared as alcohol-insoluble solids) of bush butter fruit endocarp tissue at three stages of ripeness. The amount of soluble pectins remained constant but they underwent a gradual depolymerisation during ripening. The CDTA extracts were fractionated by anion exchange and the subfractions were analysed for their sugar composition and molecular weight distribution. For all degrees of ripeness the extracts were composed of three minor peaks and two major peaks. The minor peaks appeared to be composed of xyloglucan and mannan-type polymers for the first peak and arabinogalactan-type polymers for the other two peaks. The two main peaks were retained on the column. The first was exclusively composed of homogalacturonan polymers and the second contained principally highly branched rhamnogalacturonan polymers. During ripening, both homogalacturonan and rhamnogalacturonan populations were modified. Modifications in the rhamnogalacturonan fraction were principally marked by the accumulation of low-molecular-weight rhamnoglacturonan polymers in the course of ripening. © 2001 Society of Chemical Industry [source] Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatographyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2008Guanghui Han Abstract The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe3+ immobilized metal affinity chromatography (Fe3+ -IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe3+ -IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%. [source] Serum biomarker profiling by solid-phase extraction with particle-embedded micro tips and matrix-assisted laser desorption/ionization mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2008Arti Navare One of the main challenges in high-throughput serum profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the development of proteome fractionation approaches that allow the acquisition of reproducible profiles with a maximum number of spectral features and minimum interferences from biological matrices. This study evaluates a new class of solid-phase extraction (SPE) pipette tips embedded with different chromatographic media for fractionation of model protein digests and serum samples. The materials embedded include strong anion exchange (SAX), weak cation exchange (WCX), C18, C8, C4, immobilized metal affinity chromatography (IMAC) and zirconium dioxide particles. Simple and rapid serum proteome profiling protocols based on these SPE micro tips are described and tested using a variety of MALDI matrices. We show that different types of particle-embedded SPE micro tips provide complementary information in terms of the spectral features detected for , -casein digests and control human serum samples. The effect of different sample pretreatments, such as serum dilution and ultrafiltration using molecular weight cut-off membranes, and the reproducibility observed for replicate experiments, are also evaluated. The results demonstrate the usefulness of these simple SPE tips combined with offline MALDI-TOF MS for obtaining information-rich serum profiles, resulting in a robust, versatile and reproducible open-source platform for serum biomarker discovery. Copyright © 2008 John Wiley & Sons, Ltd. [source] Determination of S -phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography-tandem mass spectrometric methodBIOMEDICAL CHROMATOGRAPHY, Issue 6-7 2006Yinghe Li Abstract S -phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S- phenylmercapturic acid in human urine. Isotope-labeled S- phenylmercapturic acid- d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S- phenylmercapturic acid (m/z 238 , 109) and S- phenylmercapturic acid - d5 (m/z 243 , 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400,200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved. Copyright © 2006 John Wiley & Sons, Ltd. [source] The influence of homogenisation conditions on biomass-adsorbent interactions during ion-exchange expanded bed adsorptionBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Jürgen J. Hubbuch Expanded bed adsorption (EBA) is an integrative step in downstream processing allowing the direct capture of target proteins from cell-containing feedstocks. Extensive co-adsorption of biomass, however, may hamper the application of this technique. The latter is especially observed at anion exchange processes as cells or cell debris are negatively charged under common anion exchange conditions. The restrictions observed under these conditions are, however, directly related to processing steps prior to fluidised bed application. In this study, it could be shown that the effective surface charge of cell debris obtained during homogenisation is closely related to the debris size and thus to the homogenisation method and conditions. The amount and thus effect of cells binding to the adsorbent could be significantly decreased when optimising the homogenisation step not only towards optimal product release but towards a reduction of debris size and charge. The lower size and charge of the debris results not only in a reduced retention probability but also, in a lower collision probability between debris and adsorbent. The applicability was shown in an example where the homogenisation conditions of E. coli were optimised towards EBA applications. In a previous report (Reichert et al., 2001) studying the suitability of EBA for the capture of formate dehydrogenate from E. coli homogenate the pseudo affinity resin Streamline Red was identified as the only suitable adsorbent. The new approach, however, led to a system where anion exchange as capture step became possible, however, to the cost of binding capacity. © 2006 Wiley Periodicals, Inc. [source] Development of a Purification Process for Adenovirus: Controlling Virus Aggregation to Improve the Clearance of Host Cell DNABIOTECHNOLOGY PROGRESS, Issue 2 2005John O. Konz The clearance of host cell DNA is a critical goal for purification process development for recombinant Ad5 (rAd5) based vaccines and gene therapy products. We have evaluated the clearance of DNA by a rAd5 purification process utilizing nuclease digestion, ultrafiltration, and anion exchange (AEX) chromatography and found residual host cell DNA to consistently reach a limiting value of about 100 pg/1011 rAd5 particles. Characterization of the purified rAd5 product using serial AEX chromatography, hydroxyapatite chromatography, or nuclease treatment with and without particle disruption showed that the residual DNA was associated with virus particles. Using a variety of additional physical characterization methods, a population of rAd5 virus in an aggregated state was detected. Aggregation was eliminated using nonionic detergents to attenuate hydrophobic interactions and sodium chloride to attenuate electrostatic interactions. After implementation of these modifications, the process was able to consistently reduce host cell DNA to levels at or below 5 pg/1011 rAd5 particles, suggesting that molecular interactions between cellular DNA and rAd5 are important determinants of process DNA clearance capability and that the co-purifying DNA was not encapsidated. [source] Ionic Liquids Made with Dimethyl Carbonate: Solvents as well as Boosted Basic Catalysts for the Michael ReactionCHEMISTRY - A EUROPEAN JOURNAL, Issue 45 2009Massimo Fabris Dr. Abstract This article describes 1),a methodology for the green synthesis of a class of methylammonium and methylphosphonium ionic liquids (ILs), 2),how to tune their acid,base properties by anion exchange, 3),complete neat-phase NMR spectroscopic characterisation of these materials and 4),their application as active organocatalysts for base-promoted carbon,carbon bond-forming reactions. Methylation of tertiary amines or phosphines with dimethyl carbonate leads to the formation of the halogen-free methyl-onium methyl carbonate salts, and these can be easily anion-exchanged to yield a range of derivatives with different melting points, solubility, acid,base properties, stability and viscosity. Treatment with water, in particular, yields bicarbonate-exchanged liquid onium salts. These proved strongly basic, enough to efficiently catalyse the Michael reaction; experiments suggest that in these systems the bicarbonate basicity is boosted by two orders of magnitude with respect to inorganic bicarbonate salts. These basic ionic liquids used in catalytic amounts are better even than traditional strong organic bases. The present work also introduces neat NMR spectroscopy of the ionic liquids as a probe for solute,solvent interactions as well as a tool for characterisation. Our studies show that high catalytic efficacy of functional ionic liquids can be achieved by integrating their green synthesis, along with a fine-tuning of their structure. Demonstrating that ionic liquid solvents can be made by a truly green procedure, and that their properties and reactivity can be tailored to the point of bridging the gap between their use as solvents and as catalysts. [source] Dynamic Equilibria in Solvent-Mediated Anion, Cation and Ligand Exchange in Transition-Metal Coordination Polymers: Solid-State Transfer or Recrystallisation?CHEMISTRY - A EUROPEAN JOURNAL, Issue 35 2009Xianjin Cui Abstract The solution properties of a series of transition-metal,ligand coordination polymers [ML(X)n], [M=AgI, ZnII, HgII and CdII; L=4,4,-bipyridine (4,4,-bipy), pyrazine (pyz), 3,4,-bipyridine (3,4,-bipy), 4-(10-(pyridin-4-yl)anthracen-9-yl)pyridine (anbp); X=NO3,, CH3COO,, CF3SO3,, Cl,, BF4,; n=1 or 2] in the presence of competing anions, metal cations and ligands have been investigated systematically. Providing that the solubility of the starting complex is sufficiently high, all the components of the coordination polymer, namely the anion, the cation and the ligand, can be exchanged on contact with a solution phase of a competing component. The solubility of coordination polymers is a key factor in the analysis of their reactivity and this solubility depends strongly on the physical properties of the solvent and on its ability to bind metal cations constituting the backbone of the coordination polymer. The degree of reversibility of these solvent-induced anion-exchange transformations is determined by the ratio of the solubility product constants for the starting and resultant complexes, which in turn depend upon the choice of solvent and the temperature. The extent of anion exchange is controlled effectively by the ratio of the concentrations of incoming ions to outgoing ions in the liquid phase and the solvation of various constituent components comprising the coordination polymer. These observations can be rationalised in terms of a dynamic equilibrium of ion exchange reactions coupled with Ostwald ripening of crystalline products. The single-crystal X-ray structures of [Ag(pyz)ClO4], (1), {[Ag(4,4,-bipy)(CF3SO3)],CH3CN}, (2), {[Ag(4,4,-bipy)(CH3CN)]ClO4, 0.5,CH3CN}, (3), metal-free anbp (4), [Ag(anbp)NO3(H2O)], (5), {[Cd(4,4,-bipy)2(H2O)2](NO3)2,4,H2O}, (6) and {[Zn(4,4,-bipy)SO4(H2O)3] ,2,H2O}, (7) are reported. [source] | |||||||||||||||||||