Home About us Contact
|
Home About us Contact | ||
|
|
||
Phase HPLC (phase + hplc)
Kinds of Phase HPLC Selected AbstractsLysozyme as Pathogen-Recognition Protein in the Hemolymph of Galleria mellonellaENTOMOLOGICAL RESEARCH, Issue 3 2003In Hee LEE ABSTRACT Recognition of invading micro-organisms into hemolymph is a pivotal event for triggering diverse immune mechanisms in insects. It has been known that this recognition was mediated by the binding of hemolymph proteins to pattern-molecules on the cell surface of microbes. Recently, I found that the lysozyme in the G. mellonella hemolymph has binding affinity to cell-walls of Gram (-), (±) bacteria and fungus (Candida albicans). After the hemolymph was incubated with heat-killed microbes and treated with acidic buffer containing high concentration of NaCl, several plasma proteins detached from microbes were detected by reverse phase HPLC and SDS-PAGE analyses. Of binding proteins, it was assumed that the major one might be a lysozyme, which was previously characterized in the G. mellonella hemolymph. Furthermore immunoblot analysis performed with antiserum to G. mellonella lysozyme revealed that it was a lysozyme. [source] Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst speciesJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2001A. Atrih Aims: The aim of this work was to compare the chemical structure of the spore cortex of a range of species, and to determine any correlation between cortex structure and spore resistance properties. Methods and Results: The fine chemical structure of the cortex of Bacillus subtilis, Bacillus megaterium, Bacillus cereus and Clostridium botulinum was examined by muropeptide analysis using reverse phase HPLC. There is a conserved basic structure between peptidoglycan of these species, with the only difference being the level of de -N -acetylation of an amino sugar. In order to determine if an alteration in cortex structure correlates with heat resistance properties, the peptidoglycan structure and properties of B. subtilis spores prepared under different conditions were compared. Peptidoglycan from spores prepared in Nutrient Broth (NB) showed reduction in single L -alanine substituted muramic acid to only 13·9% compared with 20·6% in CCY-grown spores. NB-prepared spores are also unstable, with 161-fold less heat resistance (60 min, 85°C) and 43 times less Mn2+ content than CCY-grown spores. Addition of MnCl2 to NB led to a peptidoglycan profile similar to CCY-grown spores, sevenfold more heat resistance (60 min, 85°C) and an 86-fold increase in Mn2+ content. Addition of CCY salts to NB led all parameters to be comparable with CCY-grown spore levels. Conclusions: It has been shown that peptidoglycan structure is conserved in four spore-forming bacteria. Also, spore heat resistance is multifactorial and cannot be accounted for by any single parameter. Significance and Impact of the Study: Endospores made by diverse species most likely have common mechanisms of heat resistance. However, the molecular basis for their resistance remains elusive. [source] Synthesis of tritium- and deuterium-labeled budesonideJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 1 2008Bachir Latli Abstract Tritium-labeled budesonide was prepared by the selective reduction of a double bond in the butenylenedioxy side chain using carrier-free tritium and palladium on carbon as a catalyst in absolute ethanol. Although the reduction gave a mixture of the desired product and the expected byproducts resulting from over reduction of the other double bonds in ring A, the desired tritium-labeled budesonide was easily isolated by reverse phase HPLC and with specific activity of 54,Ci/mmol. [D8]-budesonide was prepared from 16,-hydroxyprednisolone and D8 -butyraldehyde in 1,4-dioxane in the presence of perchloric acid. The isotopic enrichment was found to be more than 99,atom% D. Copyright © 2008 John Wiley & Sons, Ltd. [source] Comparative evaluation of 99mTc-ethylene bis-l-cysteine and 99mTc-ethylene bis-l-,-homocysteine during reversed phase HPLC analysis and electrophoresis at various pH conditionsJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 4 2001K.O. Mang'era Ethylene bis- L -,-homocysteine (L,L -EH) differs from ethylene bis- L -cysteine (L,L -EC) in having an extra methylene group between each pair of amine and carboxyl groups. The objective of this study was to determine the effect of the extra methylene groups on the characteristics of the complex of these compounds with technetium-99m during analysis by reversed phase HPLC and by electrophoresis at various pH values. Up to pH 5.5, 99mTc- L,L -EH exhibits a substantially longer retention time during reversed phase HPLC than 99mTc- L,L -EC, suggesting a more lipophilic character for 99mTc- L,L -EH under these conditions. On the other hand, 99mTc- L,L -EH clearly possesses a higher negative charge in the pH range 3-6.5 as shown by the markedly greater migration towards the anode in electrophoresis experiments. A rational explanation for these seemingly opposing observations can not yet be offered. Copyright © 2001 John Wiley & Sons, Ltd. [source] Mutations on N -terminal region of Taiwan cobra phospholipase A2 result in structurally distorted effectsJOURNAL OF PEPTIDE SCIENCE, Issue 8 2008Yi-Ling Chiou Abstract In the present study, three Taiwan cobra PLA2 variants were prepared by adding an extra N -terminal Met, substituting Asn-1 by Met or deleting the N -terminal heptapeptide. Recombinant PLA2 mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8-anilinonaphthalene sulfonate (ANS) were affected by N -terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N -terminal heptapeptide or with an extra N -terminal Met added as evidenced by the inability of the two variants to bind with Ba2+. Moreover, modification of Lys residues and energy transfer within the protein-ANS complex revealed that the Ca2+ -induced change in the global structure of PLA2 was different from that in N -terminal variants. Together with the fact that an ,activation network' connects the N -terminus with the active site, our data suggest that mutagenesis on the N -terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA2. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Solid-phase synthesis of a dendritic peptide related to a retinoblastoma protein fragment utilizing a combined boc- and fmoc-chemistry approachJOURNAL OF PEPTIDE SCIENCE, Issue 5 2001Vittoria Cavallaro Abstract Dendritic peptides, often presented as multiple antigen peptides (MAPs), are widely used in immunological-based fields of research, although their synthesis can be extremely challenging. In this paper, a tetrameric dendritic MAP-like presentation of the retinoblastoma protein [649-654] sequence (4RB649-654) has been prepared using solid-phase peptide synthesis (SPPS) methods. During the synthesis of this dendritic molecule, numerous modifications to the synthetic protocols were examined. These modifications included the introduction of a combination Boc- and Fmoc-chemistry approach and also the use of 1,8-diazabicyclo[5.4.0]-undec-7-ene as a Fmoc-deprotection agent. The use in combination of Boc- and Fmoc-based synthetic strategies resulted in the production of the desired peptide molecule, 4RB649-654, in high purity and acceptable yields following purification by reversed phase HPLC. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Quantification of polyphenols with potential antioxidant properties in wines using reverse phase HPLCJOURNAL OF SEPARATION SCIENCE, JSS, Issue 12 2008Neuza Paixăo Abstract A RP-HPLC method with photodiode array detection (DAD) was developed to separate, identify and quantify simultaneously the most representative phenolic compounds present in Madeira and Canary Islands wines. The optimized chromatographic method was carefully validated in terms of linearity, precision, accuracy and sensitivity. A high repeatability and a good stability of phenolics retention times (< 3%) were obtained, as well as relative peak area. Also high recoveries were achieved, over 80.3%. Polyphenols calibration curves showed a good linearity (r2 >0.994) within test ranges. Detection limits ranged between 0.03 and 11.5 ,g/mL for the different polyphenols. A good repeatability was obtained, with intra-day variations less than 7.9%. The described method was successfully applied to quantify several polyphenols in 26 samples of different kinds of wine (red, rosé and white wines) from Madeira and Canary Islands. Gallic acid was by far the most predominant acid. It represents more than 65% of all phenolics, followed by p -coumaric and caffeic acids. The major flavonoid found in Madeira wines was trans -resveratrol. In some wines, (,)-epicatechin was also found in highest amount. Canary wines were shown to be rich in gallic, caffeic and p -coumaric acids and quercetin. [source] Bioactivity of falcarinol and the influenceof processing and storage on its content in carrots (Daucus carota L)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2003Susanne L Hansen Abstract The concentration-dependent activity of the polyacetylene falcarinol ((9Z)-heptadeca-1,9-dien-4,6-diyn-3-ol), isolated from carrots, was investigated in a bioassay with primary mammary epithelial cells in collagen gels and compared with that of ,-carotene, the orange pigment in carrots. Falcarinol showed biphasic activity, having stimulatory effects between 0.01 and 0.05 µg ml,1 and inhibitory effects between 1 and 10 µg ml,1, whereas ,-carotene showed no effect in the concentration range 0.001,100 µg ml,1. The results are discussed in relation to the health-promoting effects of carrots and related vegetables. Falcarinol was quantified in the carrot cultivars Bolero, Rodelika and Fancy by analytical reverse phase HPLC, subjected to various processing and storage conditions in order to study how long-term storage, blanching, freezing and boiling influence the content of falcarinol. Long-term storage of raw carrot cubes (1 cm3) reduced the falcarinol content by almost 35%. A similar reduction was found in steam-blanched carrot cubes (1 cm3). Long-term storage at ,24 °C of steam blanched carrot cubes did not reduce the falcarinol content further. A reduction of almost 70% in the falcarinol content was found in carrot pieces boiled in water for 12 min compared with raw carrots. Copyright © 2003 Society of Chemical Industry [source] Effects of gamma irradiation on physical and chemical properties of chickpeas (Cicer arietinum)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2002John A Graham Abstract Changes in the physical and chemical properties of chickpeas gamma irradiated with 60Co at doses of 0,50,kGy were investigated. Irradiation between 0 and 20,kGy had no significant effect on the hydration capacity of the chickpeas; however, increasing the dose from 20 to 50,kGy significantly decreased the hydration capacity owing to leaching of soluble compounds from the cotyledon to the water. There was an improvement in cooking quality (defined as degree of softness) with increased irradiation. Gelatinisation properties of the starch (measured with a Rapid Visco-Analyser) exhibited a deterioration with increasing irradiation dose, while reversed phase HPLC identified a sequential decrease in low-molecular-weight protein composition with increasing irradiation dose. © 2002 Society of Chemical Industry [source] Detection of orange juice adulteration by tangelo juice using multivariate analysis of polymethoxylated flavones and carotenoidsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 4 2002Geoffrey G Pan Abstract Reverse phase HPLC has been applied to quantify levels of polymethoxylated flavones and carotenoids in orange and tangelo juices. Lower levels of sinensetin and tetramethyl- o -scutellarein and higher levels of heptamethoxyflavone and tangeretin relative to nobiletin indicated the addition of tangelo to orange juice. ,-Cryptoxanthin and its esters, identified by positive ion electrospray mass spectrometry, were present in larger amounts relative to ,-carotene in tangelo than in orange juice. Using canonical discriminant analysis, the addition of 100,g,kg,1 tangelo to orange juice can be detected. © 2002 Society of Chemical Industry [source] MS characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy , a pilot studyPROTEOMICS - CLINICAL APPLICATIONS, Issue 7 2009Mike Kienbaum Abstract Identification of proteins from apheresis samples was performed by both SDS-PAGE and 2-D gel separation of eluted proteins from staphylococcal protein A-based immunoadsorption columns (Prosorba®) followed by MS peptide mass fingerprinting and MS/MS peptide sequencing on a MALDI QIT TOF mass spectrometer. MS/MS peptide sequencing was performed in conjunction with a micro reversed phase HPLC configured with an online MALDI plate-spotting device. Apheresis treatment had been performed in three patients with longstanding therapy refractory rheumatoid arthritis. 2-D gels displayed ca. 500 spots representing proteins that were eluted from the Prosorba® columns. From 54 gels, a total of 1256 protein spots had been picked and yielded in the identification of 56 non-redundant proteins without counting isoforms. Proteins from the eluates belong to five major groups comprising (i) immunoglobulins (IgG, IgA, IgM heavy and light chains; about 40% of the spots), (ii) proteins involved in coagulation, (iii) HDL/LDL-associated proteins, (iv) proteins from the complement system, and (v) acute phase proteins. MS analysis showed that the full-length C3 complement protein had been cleaved upon complement activation, presumably on the column, such that the anaphylatoxin C3a was produced and released during therapy. Our results are consistent with clinical observations on both patient responses to therapy and reported adverse events. For the first time, direct molecular information has become available to support mechanistic reasoning for the principle of function of staphylococcal protein A-based immunoadsorption therapy and for the explanation of adverse events. According to our results, removal and/or modulation of immune complexes together with complement activation can be regarded as the major events that are taking place during Prosorba® therapy. In order to avoid complement activation and induction of an inflammatory cascade, we suggest the prevention of C3a anaphylatoxin-related reactions during immunoadsorption therapy. [source] Liquid chromatography ion trap mass spectrometric analysis of oligosaccharides using permethylated derivativesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2001Jeannine Delaney Reversed phase liquid chromatography was combined with the multiple stage mass analysis capability of an ion trap mass spectrometer for the characterization of permethylated oligosaccharide mixtures. The new method was used to separate the components of an unlabeled permethylated maltooligomer ladder, a 2-aminobenzamide-labeled (2-AB) maltooligomer ladder, a complex mixture of 2AB-labeled bi- (B), tri- (T), and tetraantennary (Q) standards, and a mixture of recombinant glycoprotein carbohydrates from soluble CD4 with varying sialic acid (S) content. Using reversed phase HPLC, permethylated mixture components including , and , anomers were separated based on their structures. Fluorescent labeling with 2-aminobenzamide prior to permethylation was employed for off-line method development, but was not necessarily required for mass spectral analysis, as permethylation alone improved the ionization and fragmentation characteristics of the molecules. Antennae composition of permethylated derivatives was determined in MS2 where the fragmentation patterns of the Y- and B-ion series predominated, and then further evaluated in MS3, which provided additional information on branching obtained from A and X cross-ring fragmentation. Copyright © 2001 John Wiley & Sons, Ltd. [source] Enzyme-Catalysed Synthesis and Absolute Configuration Assignments of cis -Dihydrodiol Metabolites from 1,4-Disubstituted BenzenesCHEMISTRY - A EUROPEAN JOURNAL, Issue 20 2007Derek Abstract A series of ten cis -dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H,NMR spectroscopy. Absolute configurations of the majority of cis -dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis -dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples. [source] Enantioseparation of novel chiral tetrahedral clusters on an amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase by normal phase HPLCCHINESE JOURNAL OF CHEMISTRY, Issue 10 2004Wen-Zhi Li Abstract Amylose tris(3,5-dimethylphenylcamate) (ADMPC) coated on a kind of small particle silica gel was prepared. On this ADMPC chiral stationary phase (CSP), the direct enantiomeric separation of six novel chiral transition metal tetrahedral clusters has firstly been achieved using n -hexane as the mobile phase containing various alcohols as modifiers. The effect of mobile phase modifiers and the structural variation of the solutes on their retention factors (k) and resolutions (Rs) were investigated. The result suggests that not only the structure and concentration of alcohol in mobile phase, but also the structural differences in racemates can have a pronounced effect on enantiomeric separation. ADMPC-CSP is a suitable CSP for the optical resolution of chiral tetrahedral cluster by HPLC. [source] | |||||||||||||