bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enantiomer separation of disopyramide with capillary electrophoresis using various cyclodextrins
Authors: Juvancz Zoltán , Markides Karin E. , Jicsinszky László 
Published: May 2005

AbstractEnantiomers of disopyramide display different biological actions, and therefore chiral selective analysis is necessary. Fifteen different cyclodextrins (CDs) and CD derivatives were tested as capillary electrophoresis (CE) additives for the chiral separation of disopyramide. Eleven types of CDs showed chiral recognition features and four types had a baseline or close to baseline separation. The best resolution (Rs = 3.0) was with 15 mM carboxymethylated β‐CD (pH 4.9). A sharp decrease in the selectivity of γ‐phosphate (γ‐PhoCD) was observed in the pH range of 2–3, indicating a structural change of γ‐PhoCD. The enantiomers of disopyramide were separated in its ionized as well as neutral forms using acidic substituted CDs. The results show that the size of the CD cavity can not be used as a guide to estimate chiral separations, suggesting a more complex separation mechanism of these CDs towars disopyramide.
Enantioselective determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in serum, urine and hair by capillary electrophoresis
Authors: Frost Michael , Köhler Helga , Blaschke Gottfried 
Published: May 2005

AbstractA capillary electrophoresis method has been developed for the enantioselective determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in serum and urine. After addition of the internal standard diphenhydramine, the serum and urine samples were extracted at pH 9–10 withn‐hexane. Different cyclodextrins were tested for use as chiral selectors. The best results for simultaneous separation of methadone and EDDP were obtained using heptakis‐(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DIMEB). Data on the accuracy and precision of the method are presented. The method was applied to serum, urine, and hair samples, from individuals undergoing methadone therapy, under consideration of multi‐drug abuse.
Separation of tumor necrosis factor α isoforms by two‐dimensional polyacrylamide gel electrophoresis
Authors: Watts Alan D. , Hunt Nicholas H. , Hambly Brett D. , Chaudhri Geeta 
Published: May 2005

AbstractThe mouse macrophage cell‐line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose‐dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two‐dimensional (2‐D) electrophoresis with immobilised pH gradient 3–10 linear gels as the first dimension. After radiolabelling of cells with 35S, both cell‐associated and supernate‐associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2‐D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.
Polymorphism of 5′ of the catalase gene in Hungarian acatalasemia and hypocatalasemia
Authors: Góth László , Vitai Márta 
Published: May 2005

AbstractThe amplified fragment length polymorphism of Hinfl on the promoter region of the catalase gene in Hungarian acatalasemic and hypocatalasemic patients yielded three different patterns with five bands in total. The sequence analyses revealed A‐to‐T, C‐to‐A, and C‐to‐T mutations at positions −21, −20, and −18 upstream of the translational initiation site. The −21 A‐to‐T mutations were more frequent in acatalasemic and hypocatalasemic patients (36/2) than in controls (18/14). This mutation had been detected in Japanese acatalasemic patients while the other two are novel mutations. Two extra bands in the Hinfl pattern are due to star‐like activity that cleaved a G/ATTT sequence at position −4 to 0 upstream of the initiation site.
Cellular electrophoretic mobility data: A first approach to a database
Authors: Slivinsky George G. , Hymer Wesley C. , Bauer Johann , Morrison Dennis R. 
Published: May 2005

AbstractCellular electrophoretic mobility values of 288 types of eucaryotic cells were collected from literature published worldwide by a series of authors during the past forty years and arranged in a list. This list contains well‐known recent electrophoretic results and also data that cannot be found anymore with modern literature retrieval systems. It will be a valuable help for scientists trying to purify cell populations. In addition, it confirms the observation that most eucaryotic cells have very similar electrophoretic mobilities, ranging from 40% above to 50% below the electrophoretic mobility of human erythrocytes, and thus reinforces the suggestion that electrophoretic mobilities of eucaryotic cells are subjected to strong biological controls.
The resolution between two native proteins and between their sodium dodecyl sulfate‐complexes in agarose and polyacrylamide gel electrophoresis
Authors: Chen Nong , Chrambach Andreas 
Published: May 2005

AbstractCommercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE‐1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG‐, SeaKem Gold‐agarose, 2% N,N′‐methylenebisacrylamide cross‐linked polyacrylamide, MetaPhor‐XR‐, and SeaPrep‐agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4–6% SeaPrep agarose than in 4–6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS‐proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards — proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3–8%, and that therefore the molecular “fit” in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius (“fit”) is also suggested by the fact that the two SDS‐proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.
Capillary electrophoresis of pesticides: V. analysis of pyrethroid insecticides via their hydrolysis products labeled with a fluorescing and UV absorbing tag for laser‐induced fluorescence and UV detection
Authors: Karcher Arron , El Rassi Ziad 
Published: May 2005

AbstractSome representative standard pyrethroid insecticides, namely permethrin, phenotrin, cypermethrin, sanmarton and fenpropathrin, were subjected to base hydrolysis with the aim of facilitating the indirect determination of these neutral species of low water solubilities by aqueous capillary electrophoresis. This first involved the base fragmentation of the pyrethroids in alcohol buffer (pH 12.0), and then the selective tagging of the carboxylated hydrolytic products with 7‐aminonaphthalene‐1,3‐disulfonic acid (ANDSA) via a condensation reaction in the presence of organic soluble carbodiimide. The tagging of the hydrolytic products with ANDSA imparted each of the derivatives with two strong sulfonic acid groups whose permanent charges were necessary for achieving aqueous capillary electrophoresis. In addition, the labeling with ANDSA allowed the detection of the derivatives at low levels by capillary electrophoresis laser‐induced fluorescence. The geometric and optical isomers of the ANDSA derivatives of the pyrethroid hydrolytic products were best separated when using electrolyte systems composed of sodium phosphate buffer, pH 6.5, containing n‐octylglucoside chiral surfactant in the presence of small amounts of acetonitrile (e.g., 10% v/v).
From genome to proteome: Protein map of Haemophilus influenzae
Authors: Langen Hanno , Gray Chris , Röder Daniel , Juranville Jean‐François , Takacs Béla , Fountoulakis Michael 
Published: May 2005

AbstractHigh‐resolution two‐dimensional (2‐D) polyacrylamide gel electrophoresis allows the separation of complex biological mixtures (i.e., several hundred proteins from a bacterial cell lysate) in a single experiment. In this report proteins from Haemophilus influenzae were separated by 2‐D gels and analyzed by peptide mass fingerprinting and/or amino acid analysis. By comparing the peptide mass profiles and the amino acid composition with the Haemophilus influenzae database, 119 protein spots were identified. The combination of amino acid analysis and peptide mass fingerprinting is a powerful tool for a rapid and economical identification of a large number of proteins resolved by 2‐D gels. Studies on gene regulation and changes of protein expression upon drug treatment require quick and serial analysis techniques to efficiently identify potential new drug targets.
Two‐dimensional map of Haemophilus influenzae following protein enrichment by heparin chromatography
Authors: Fountoulakis Michael , Langen Hanno , Evers Stefan , Gray Chris , Takács Béla 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis separates several hundred protein molecules in one single experiment and is efficiently used to study the products expressed by different genomes. Low‐copy‐number gene products are invisible on a stained two‐dimensional map and must be enriched such that sufficient amounts are present for visualization and identification. We investigated the enrichment of proteins of the bacterium Haemophilus influenzae by chromatography on immobilized heparin which has affinity for growth and protein biosynthesis factors. Total soluble proteins of the microorganism were fractionated on Heparin‐Actigel which resulted in enrichment of approximately 160 proteins. The eluates, representing about 40% of the applied proteins, were analyzed by two‐dimensional gel electrophoresis and the protein spots were characterized by amino acid composition analysis and matrix‐assisted laser desorption ionization mass spectrometry. The proteins enriched by chromatography on the heparin gel were not exclusively low‐copy‐number gene products and they did not exclusively belong to one single class of proteins. The proteins that bound to the heparin gel are indicated in a two‐dimensional protein map which includes more than 110 newly identified proteins.
Identifying the major proteome components of Haemophilus influenzae type‐strain NCTC 8143
Authors: Link Andrew J. , Hays Lara G. , Carmack Edwin B. , Yates John R. 
Published: May 2005

AbstractWith the completion of the Haemophilus influenzae Rd genomic sequence, we know the identity of most of the theoretical proteins in the proteome of this bacterium. However, the most abundant components of the actual proteome are unknown. Using mass spectrometry and two‐dimensional gel electrophoresis (2‐DE), we sequenced and analyzed the most abundant proteins observed in the ATCC reference strain of H. influenzae, NCTC 8143 (303 of ≈︁ 400 Coomassie‐stained 2‐DE spots). To automate the identification of 2‐DE spots, we coupled a liquid autosampler to a microcolumn liquid chromatography electrospray ionization tandem mass spectrometer capable of identifying 22 spots per day. From the 303 sequenced spots, we identified 263 unique proteins. Most of the abundant proteins lie in an isoelectric point range of pH 4–7 and a molecular mass range of 10–100 kDa. Of the observed proteins, the most abundant is the outer membrane protein P2. Based on variety and abundance, proteins involved in energy metabolism and macromolecular synthesis are the dominant classes of proteins. Unexpectedly, tryptophanase was identified as a highly abundant protein in the strain NCTC 8143 whose sequence is rot present in the genome of the Rd strain. By searching the tandem mass spectra against the translated genomic sequence, we identified several proteins which were not annotated in the genomic sequence. Surprisingly, 22% of the identified 2‐DE spots represent isoforms in which gene products with the same primary sequence have different observed pI and Mr, indicating that these proteins are post‐translationally processed. Although most proteins' predicted and observed isoelectric points and molecular masses show reasonable concordance, the observed values for several proteins deviate significantly from the predicted values. These anomalies may represent either highly processed proteins or misinterpretations of the genomic sequence. Using the technology developed in this project, the protein expression of other strains of H. influenzae grown under different environmental conditions can be compared to identify differences in their proteomes.
Peptide‐mass fingerprinting and the ideal covering set for protein characterisation
Authors: Wise Michael J. , Littlejohn Timothy G. , Humphery‐Smith Ian 
Published: May 2005

AbstractThe rules that govern the dynamics of protein characterisation by peptide‐mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo‐endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu‐C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of > 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52 908 Da to 258 314 Da, while the failure rate for a single cutter in identifying database entries varied from 10 865 (8.4%) to 23 290 (18.1 %). PMF is likely to be a mainstay of any high‐throughput protein screening strategy for large‐scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.
Evaluation of algorithms used for cross‐species proteome characterisation
Authors: Cordwell Stuart J. , Humphery‐Smith Ian 
Published: May 2005

AbstractThe ability to effectively search databases for the identification of protein spots from two‐dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N‐ and C‐terminal microsequence, amino acid composition, and peptide‐mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty‐four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7–100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non‐sequence‐dependent software in proteome analysis. The data obtained also predicted that ‘sequence tagging’ of peptide fingerprints would need to be accompanied by at least 11–20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.
Identification of procaryotic developmental stages by statistical analyses of two‐dimensional gel patterns
Authors: Vohradsky Jiří , Li Xin‐Ming , Thompson Charles J. 
Published: May 2005

AbstractMultivariate statistical comparisons of two‐dimensional protein (2‐D) gel patterns were used for the first time to define stages of a biological developmental system. The differentiating procaryote, Streptomyces coelicolor, was radiolabeled in liquid cultures at 16 intervals during development, and radioactive proteins were separated and quantified on 2‐D gels. Cluster, principal component, and correlation analyses classified these gel patterns into four distinct groups, each reflecting a pattern of gene expression specific for a stage of development. These studies focused our attention on a phase of arrested growth as a key regulatory transition leading to secondary metabolism and a phase of renewed growth. Proteins whose synthesis was switched on or off during the “transitional” phase (some 21 and 18, respectively) were identified and will be the focus of future studies designed to identify their physiological or regulatory function.
First steps from a two‐dimensional protein index towards a response‐regulation map for Bacillus subtilis
Authors: Antelmann Haike , Bernhardt Jörg , Schmid Roland , Mach Hiltraut , Völker Uwe , Hecker Michael 
Published: May 2005

AbstractData on the identification of proteins of Bacillus subtilis on two‐dimensional (2‐D) gels as well as their regulation are summarized and the identification of 56 protein spots is included. The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt‐ and ethanol stress as well as oxidative stress was analyzed. N‐terminal sequencing of protein spots allowed the identification of 93 proteins on 2‐D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions. A computer‐aided analysis of the 2‐D gels was used to monitor the synthesis profile of more than 130 protein spots. Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entrely different stimuli. The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B. subtilis. These investigations demonstrated that the alternative sigma factor σB is involved in the regulation of almost all of the general stress proteins and that the phoPR two‐component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation.
Hybridization capture of microsatellites directly from genomic DNA
Authors: Refseth Unn Hilde , Fangan Bjørn Magne , Jakobsen Kjetill Sigurd 
Published: May 2005

AbstractA rapid approach for isolation of microsatellites and other tandem repeated sequences in described. The method is based on hybridization capture of repetitive elements from digested genomic DNA using biotinylated oligonucleotide probes in solution and subsequent attachment to magnetic beads coated with streptavidin. Captured fragments are amplified by adapter polymerase chain reaction (PCR) and the PCR products enriched for microsatellites cloned directly into a T‐vector for sequencing. The results presented here show that this approach is highly effective, allowing di‐ and trinucleotide repeats to be isolated and sequenced directly from fish and mammalian genomic DNA within four to five days. Assuming a density and relative abundance of repeats with AC/GT motifs corresponding to that found in the human genome, the protocol presented gives at least a 35‐fold enrichment of AC/GT microsatellites using an (AC)10 oligo probe. In addition, four out of five sequences captured by a (CAG)9 oligo probe contained one or several CAG repeat arrays. The efficiency of this direct approach suggests that it can be used for extracting other types of tandem and interspersed repeated sequences (including transposons, rRNA and tRNA genes and proviruses) from vertebrate genomes.