bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional lectin affinity electrophoresis of α‐fetoprotein: Characterization of erythroagglutinating phytohemagglutinin‐dependent microheterogeneity forms
Authors: Taketa Kazuhisa , Ichikawa Eriko , Sato Jiro , Taga Hiroko , Hirai Hidematsu 
Published: May 2005

AbstractBy means of two‐dimensional lectin affinity electrophoresis of human α‐fetoprotein (AFP) from different sources, AFP bands separated with erythroagglutinating phytohemagglutinin (E‐PHA) were further characterized with other lectins of known oligosaccharide specificities. The results with a cord serum AFP revealed that not only AFP‐P2 (E‐PHA‐nonreactive) but also AFP‐P4 and P5 (E‐PHA‐reactive) had affinities for Concanavalin A (Con A) and Allomyrina dichotoma lectin (allo A), indicating that the cord serum AFP has nonbisected biantennary complex‐type oligosaccharides with the terminal galactose on Man α1 → 6 residue sialylated at the C‐6, but not C‐3, position. On the other hand, the results with a hepatoblastoma (HUH‐6 C1–5 cell line) AFP showed that not only AFP‐P5 but also AFP‐P1 (E‐PHA‐nonreactive) and P3 (E‐PHA‐less reactive) had Con A‐nonreactive AFP and that AFP‐P1 had AFP‐A1 (allo A‐nonreactive) and AFP‐A2 (allo A‐less reactive), and AFP‐P3 and P4 had AFP‐A1s (allo A‐nonreactive), as main components, in addition to the spots of cord serum AFP. Most of the E‐PHA‐dependent bands of AFP were further subdivided with Lens culinaris agglutinin (LCA‐A) into LCA‐A‐reactive, weakly reactive and nonreactive spots. Similar results were obtained with AFP preparations from hepatocullular carcinomas and other malignancies, indicating that the bisected bi‐(or tri‐and tetra‐) antennary sugar chains with the exposed terminal galactose of the Man α 1 → 6 arm as well as those with the C‐3 sialylated galactose residues could be expressed in AFP upon malignant transformation. The two‐dimensional lectin affinity electrophoresis thus proves useful for further characterization of lectin‐dependent AFP bands.
Crossed affino‐immunoelectrophoresis or affino‐blotting with lectins: Advantages and limitations for glycoprotein studies
Authors: Faye Loïc , Salier Jean‐Philippe 
Published: May 2005

AbstractIn contrast to the conventional combination of physical, chemical and enzymatic methods used for a structural analysis of glycans in glycoproteins, alternative methods involve affinity electrophoresis as a tool for the detection, characterization, and quantitation of glycoproteins and their carbohydrate moiety, owing to interactions with lectins. Two major approaches involve (i) crossed affino‐immunoelectrophoresis and variations thereof, whereby lectin/glycoprotein interactions occur during the electrophoretic runs, or (ii) affino‐blotting, where the glycoproteins are electrophoretically separated and then immobilized onto a solid support prior to their interaction with lectins. A critical comparison of these two series of techniques is the scope of the present paper. These techniques are of high interest by virtue of their ability at differentiating a classical glycan structure from unusual oligosaccharide side chains. The former structures will usually be qualitatively and quantitatively described with the easy and fast procedures as well as the simple equipment required for crossed affino‐immunoelectrophoresis or affino‐blotting, whereas the latter will be good candidates for further structural analyses.
Enzyme activity electrophoresis: Development and applications
Authors: Poulsen Otto M. , Jacobsen Tomas , Hau Jann , Jensen Bo , Vodder Lise , Andersen Frantz 
Published: May 2005

AbstractThe development of rocket enzyme activity electrophoresis for the detection and quantification of various proteinases, lipases and pectinases is presented. Rocket enzyme activity electrophoresis is more sensitive than the radial diffusion assay and often enables distinction between qualitatively different enzymes present in the same samples, whereas the radial diffusion assay only provides information on the overall enzyme activity. However, calibration and optimization of the enzyme activity electrophoretic assay have to be performed for each new enzyme‐substrate system to be analyzed. Some of the common pitfalls in the development of new enzyme activity electrophoretic assays are presented. Enzyme activity electrophoresis can be applied in combination with other electrophoretic assays. Particularly the combination of enzyme activity electrophoresis with various immunoelectrophoretic methods can provide detailed information on the enzymes studies.
Free‐flow electrophoresis under microgravity: Evidence for enhanced resolution of cell separation
Authors: Hannig Kurt , Kowalski Matthias , Klöck Gerd , Zimmermann Ulrich , Mang Volker 
Published: May 2005

AbstractA mixture of fixed rabbit, guinea pig and rat erythocytes, suspended in a relatively conductive solution, was separated by means of continuous free flow electrophoresis (CFFE) under 1 g ‐ and μg ‐ conditions using a specially designed electrophoretic module. Short duration μg conditions were realized on board a sounding rocket. Due to the energy input and the associated thermal convection a separation of the three differently charged cell types in distinct peaks was not possible under 1 g‐conditions as shown by reference experiments on the ground before launch. In contrast to the poor resolution under 1 g‐conditions, clear separation of the cell mixture could be recorded after lift‐off of the rocket under μg‐conditions. Repeated measurements demonstrated that the separation profile was completely stable during the entire μg‐phase of about 6 min. Since the CFFE experiment in space was an exact replica of the ground reference experiments, the results demonstrated unambiguously the potential of CFFE for cell separation under μg‐conditions in media of high ionic strength.
Electrophoretic transport of solutes in aqueous two‐phase systems
Authors: Levine Mark L. , Bier Milan 
Published: May 2005

AbstractElectrophoretic transport of proteins across the interface between the phases of an aqueous polymer two‐phase system can be greatly impeded in comparison with transport within the individual phase. This effect can be controlled by modifying the affinity of the protein for a phase by suitable manipulations of such variables as pH. The effect is not caused by differences in the electrophoretic velocity between the two phases, nor by large changes in pH at the interface. An analogy exists between this phenomenon and the related subject of diffusion of electrolytes across the phase interface.
Enzyme visualization with partially dehydrated agarose substrate layers: Detection of paraoxonase after thin‐layer isoelectric focusing in agarose
Authors: Haas Arved , Mallinckrodt Marika Geldmacher‐Von 
Published: May 2005

AbstractA new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme visualization is performed by a modified sandwich procedure which is faster and also affords higher resolution and considerably improved sensitivity. Up to seven paraoxon splitting isoenzymes can be visualized and clearly distinguished from arylesterases and phosphatases by using 3‐naphtyl acetate, paraoxon, 5‐bromo‐4‐chloro‐3‐indolyl acetate and 5‐bromo‐4‐chloro‐3‐indolyl phosphate as substrates. The new technique is also able to differentiate between paraoxonase isoenzymes sensitive to EDTA and those which are EDTA‐stable. Immunofication with anti‐human serum albumin‐antibodies revealed similar isoelectric points for these isoenzymes, although they are not assumed to be identical. The new technique may prove useful in other applications of enzyme visualization where diffusion of enzymes and /or cleavage products is the major problem.
Resolution of a paradox in the electrophoresis of DNA in agarose gels
Authors: Stellwagen Nancy C. , Holmes Diana L. 
Published: May 2005

AbstractA paradox was observed in a previous study of the electrophoresis of linear DNA fragments in agarose gels (D. L. Holmes and N. C. Stellwagen, Electrophoresis 1990, 11, 5–15). The pore size of the agarose matrix was more accurately determined if the root‐mean‐square radius of gyration was used to measure DNA macromolecular size. However, the Ogston equations were obeyed and other gel parameters such as the apparent fiber radius and fiber volume appeared to be better described if the geometric mean radius was used to measure DNA size. This paradox can be resolved if relative mobilities (with respect to the smallest DNA molecule in the data set) are used to construct the Ferguson piots, instead of absolute mobilities. Using relative mobilities and the root‐mean‐square radius of gyration, the Ogston equations are obeyed and the pore size of the matrix is consistent with values determined by other methods.
Two‐dimensional gel protein database of Saccharomyces cerevisiae
Authors: Boucherie Hélian , Sagliocco Francis , Joubert Richard , Maillet Isabelle , Labarre Jean , Perrot Michel 
Published: May 2005

AbstractWith the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced. One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing. Another is to understand how all yeast genes act in concert to ensure and maintain cell organization. Two‐dimensional (2‐D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression. In prospect of these studies we have undertaken the construction of a yeast 2‐D gel protein database that contains information on polypeptides of the yeast protein map. In this paper we report the information presently contained in this database. The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N‐terminal acetylated proteins, mitochondrial proteins, glucose‐repressed proteins, heat shock induced proteins and proteins encoded by intron‐containing genes. In all, 600 spots are annotated. These data can be accessed on the Yeast Protein Map server through the World Wide Web network.
cDNA expression and human two‐dimensional gel protein databases: Towards integrating DNA and protein information
Authors: Leffers Henrik , Dejgaard Kurt , Honoré Bent , Madsen Peder , Nielsen Morten S. , Celis Julio E. 
Published: May 2005

AbstractThe rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two‐dimensional (2‐D) gel electrophoresis in combination with computer‐aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To data, comprehensive 2‐D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http://biobase.dk/cgi‐bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2‐D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post‐translational trimmings and modifications of the proteins. The method, in combination with 2‐D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.
Analysis of chaperonin‐containing TCP‐1 subunits in the human keratinocyte two‐dimensional protein database: Further characterisation of antibodies to individual subunits
Authors: Hynes Gillian , Celis Julio E. , Lewis Victoria A. , Carne Alexander , U Sally , Lauridsen Jette B. , Willison Keith R. 
Published: May 2005

AbstractThe chaperonin‐containing TCP‐1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research. CCT consists of at least eight different subunit types encoded by independent but related genes, and a set of antibodies that recognise individual subunits has proved useful in the characterisation and functional analysis of CCT. These antibodies were used to identify subunits of CCT in the human keratinocyte two‐dimensional protein database. Accurate values for the pI and molecular mass of human CCT subunits were determined from the database, and biological data was obtained regarding changes in subunit levels in response to extracellular agents and growth conditions. The second part of the study describes the characterisation of seven monoclonal antibodies raised against mouse TCP‐1, also known as CCTα, using a combination of epitope mapping and immunoblot analysis of protein extracts from different species and tissue types. Some antibodies were not monospecific for TCP‐1, and a number of epitope‐related proteins were identified.
The use of two‐dimensional gel electrophoresis in studies on the role of cytoskeletal plaque proteins as tumor suppressors
Authors: Ben‐Ze'ev Avri 
Published: May 2005

AbstractThe morphology and functions of cell sand tissues are determined, in a large part, by mechanical forces generated at cell‐cell and cell‐extracellular matrix (ECM) contacts. At these sites, transmembrane adhesion receptors of the integrin and cadherin families are linked, via their cytoplasmic domain, to the cytoskeleton by submembranal plaque proteins such as vinculin, α‐actinin and the cell‐cell junctional plaque proteins α‐ and β‐catenin and plakoglobin (or γ‐catenin). Recent studies have implicated this link of structural molecules between the outside and inside of the cell in signal transduction. We have shown that the expression of junctional plaque proteins is modulated during growth stimulation and differentiation, and is dramatically reduced in certain tumor cells. To study the functional significance of these changes in expression, we have used recombinant DNA technologies to overexpress or suppress the levels of junctional plaque proteins. In addition, we eliminated the expression of vinculin in embryonal stem (ES) cells and in the embryonal carcinoma F9 line by gene disruption employing homologous recombination. The results have indicated that moderate overexpression of cell‐ECM plaque proteins results in reduced cell motility. In contrast, suppression of their expression, by antisense transfection, led to enhanced motility and conferred anchorage independent growth and tumorigenicity, upon injection into nude mice. These findings suggest that submembranal plaque proteins can act as effective tumor suppressors. In agreement with this notion, we found in several tumor cell lines diminished levels of junctional plaque proteins. Restoration of their level to that found in normal cells resulted in tumor suppression after their injection into experimental animals. Here we demonstrate the usefulness of the application of two dimensional (2‐D) gel electrophoresis in these studies.
Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications
Authors: Wätzig Hermann , Degenhardt Matthias , Kunkel Annette 
Published: May 2005

AbstractThis review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electrosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.
Analyte identification in capillary electrophoretic separation techniques
Authors: Kok Steven J. , Velthorst Nel H. , Gooijer Cees , Th. Brinkman Udo A. 
Published: May 2005

AbstractA review on applications of on‐line hyphenation in capillary electrophoresis and capillary electrochromatography for the identification of migrating analytes is presented. There is an urgent need for unambiguous analyte identification by combining spectral information and observed migration times, because the parameters influencing the migration times and separation efficiencies in these separation techniques are not easily controlled, especially when real samples containing unknown interferences have to be analyzed. The spectrometric techniques covered here are ultraviolet and visible radiation (UV/Vis) absorption, fluorescence including fluorescence line‐narrowing spectroscopy, Raman spectroscopy, nuclear magnetic resonance and mass spectrometry. Attention is essentially confined to literature reports in which the extra information provided by the detector is really used for identification purposes, especially in real‐life samples, while the interfacing as such and analyte detectabilities in standard solutions are only briefly discussed. This article covers an extensive fraction of the literature published on this topic until the beginning of 1998.
Drug analysis by capillary electrophoresis and laser‐induced fluorescence
Authors: Couderc François , Caussé Elizabeth , Bayle Christophe 
Published: May 2005

AbstractThis review briefly presents the different laser‐induced fluorescence detectors, outlines the different dyes used to derivatize molecules which are used with capillary electrophoresis/laser‐induced fluorescence (CE‐LIF), and provides an overview and current status of CE‐LIF in drug analysis.
Separation of chiral compounds by capillary electrophoresis
Authors: Verleysen Katleen , Sandra Pat 
Published: May 2005

AbstractThis review presents the different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers. The use of charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids is discussed in detail. Neutral native and derivatized cyclodextrins are not treated because several review articles have already been published on this topic. Recent developments like the application of two chiral selectors in the same background electrolyte are highlighted.
Method development strategies for the enantioseparation of drugs by capillary electrophoresis using cyclodextrins as chiral additives
Authors: Fillet Marianne , Hubert Philippe , Crommen Jacques 
Published: May 2005

AbstractGeneral strategies for the development of capillary electrophoretic methods for the enantiomeric separation of basic, acidic or neutral drugs were developed. For all kinds of compounds, the use of a buffer made of 100 mM phosphoric acid adjusted to pH 3 with triethanolamine and containing anionic and/or uncharged cyclodextrin (CD) derivatives as chiral selectors was recommended. Two different optimization schemes depending on the acidic or basic character of the analytes, were elaborated. For most basic compounds present in cationic form at pH 3, enantiomeric separation could be achieved in the normal polarity mode. Different β‐cyclodextrin derivatives were first tested at a given concentration. Five derivatives were found to be particularly useful for enantioseparations in capillary electrophoresis (CE): the anionic carboxymethyl‐β‐CD (CMCD) and sulfobutyl‐β‐CD (SBCD) and the neutral dimethyl‐β‐CD (DMCD), trimethyl‐β‐CD (TMCD) and hydroxypropyl‐β‐CD (HPCD). After selection of the most suitable CD, its concentration was optimized with respect to chiral resolution. If necessary, a further improvement in resolution could often be obtained for the enantiomers of cationic solutes by increasing the buffer pH from 3 to 5 using CMCD as chiral additive. Another possible alternative for enhancement in chiral resolution was the addition of methanol or cyclohexanol to the buffer. For acidic drugs, essentially present in uncharged form at pH 3, and for neutral solutes, anionic CD derivatives such as SBCD or CMCD were first tested at a given concentration in the reversed polarity mode. Dual systems, based on the simultaneous addition of a charged CD (SBCD or CMCD) and a neutral CD (TMCD or DMCD), could then be investigated for resolution improvement. After optimization of the CD concentrations, the use of dual systems with CMCD at pH 5 could also be tested if necessary, especially for very weak acidic and neutral drugs. By applying these optimization strategies, 48 of the 50 drugs examined as model compounds could be fully enantioseparated by CE in short analysis times (usually less than 10 min).
Capillary electrophoresis for therapeutic drug monitoring
Authors: Brunner Lane J. , Dipiro Joseph T. 
Published: May 2005

AbstractTherapeutic drug monitoring is commonly used in both the ambulatory and hospital patient care settings. Routine measurement of concentrations of therapeutic agents in biological fluids is critical for certain drugs to maintain therapeutic benefit with minimizing drug‐associated toxicities. Many analytical laboratory techniques are currently available to measure drug concentrations in biological samples. Recently there has been an increased interest in the use of capillary electrophoresis (CE) for measuring concentrations of therapeutic drugs in patient samples. However, while there are numerous reports of CE being used to measure drug concentrations in solution and pharmaceutical dosage forms, there are relatively few reports of the use of CE for measuring therapeutic agents in patient samples. The purpose of this paper is to provide an overview of methods currently used to measure therapeutic drugs in patient samples along with possible future trends for the use of CE in therapeutic drug monitoring.