bluuebunny/crossref_metadata_2025_split huggingface dataset:
A new buffering system and its use in electrophoresis and isoelectric focusing
Authors: Bier Milan , Ostrem James , Marquez Rodolfo B. 
Published: May 2005

AbstractA novel buffer system with rather unique properties particularly well suited for preparative isoelectric focusing is described. The system was developed in response to the long standing need for pH gradients formulated using inexpensive components of known chemical composition. The system is binary consisting of buffer pairs covering the pH range of 3 to 10, in increments of less than 1 pH. The components are all readily available, nontoxic, and biologically acceptable, and are selected on the basis of their dissociation constants. The requirement for each buffer pair is that there be an overlapping pH zone where both components are largely non‐ionized, having a ratio of neutral species to total buffer concentration between about 0.90 and 0.99. This ratio is defined as the ‘electrophoretic reserve capacity’ (ERC). Within the above ERC range a stable pH gradient is obtained, assuring sharply resolved protein focusing. Buffer parameters, mainly pH, buffering capacity, ERC, transport numbers, and conductiviy, can be easily calculated and the buffer composition adjusted to meet requirements. All components are used at high concentration, typically of the order of up to 100 mM, yielding buffering capacities and conductivities analog to those customary in electrophoresis.
Analysis of scaling methods to minimize experimental variations in two‐dimensional electrophoresis quantitative data: Application to the comparison of maize inbred lines
Authors: Burstin Judith , Zivy Michel , de Vienne Dominique , Damerval Catherine 
Published: May 2005

AbstractThe analysis of two‐dimensional (2‐D) electrophoresis quantitative data from a design involving 21 maize genotypes revealed a significant experimental variation. In order to minimize this variation, we investigated the possible causes and found that it was essentially due to global effects, affecting all the spots in a gel in a similar way, and occurring during the 2‐D run/staining procedure. Three scaling methods to discard these experimental variations were, analyzed: the linear scaling method, a method based on principal component analyis, and a combined method that unites the advantages of both of the former. Comparing these three methods, we found that they led to consistent results with regard to the factor under study, i.e. the genetic factor in our case. However, the combined scaling method was the most efficient in reducing experimental variations.
SWISS‐2DPAGE: A database of two‐dimensional gel electrophoresis images
Authors: Appel Ron D. , Sanchez Jean‐Charles , Bairoch Amos , Golaz Olivier , Miu Marina , Vargas J. Reynaldo , Hochstrasser Denis F. 
Published: May 2005

AbstractThis publication presents the SWISS‐2DPAGE database which gathers data on proteins identified on various two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) maps. Each SWISS‐2DPAGE entry contains data on one protein, including mapping procedures, physiological and pathological data and bibliographical references, as well as several 2‐D PAGE images showing the protein location. Links are also provided to other databases such as SWISS‐PROT, EMBL, PROSITE and OMIM. The database has been set up on a server which may be accessed from any computer connected to the internet and it also makes it possible to display the theoretical location of proteins, the positions of which are not yet known on the 2‐D PAGE.
Application of two‐dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors
Authors: Hansen Klaus , Møller Jesper V. 
Published: May 2005

AbstractThe technique of two‐dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)‐induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [γ32P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine‐specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X‐114 fractionation, immunoreaction with specific antibodies, and comigration with 35S‐labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well‐recognized substrates (calpactin II, ezrin, EGF receptor) included β‐tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two‐dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with cell proliferation.
Degradation of fibrinogen and fibrin by plasmin and nonplasmin proteases in the chronic subdural hematoma: Evaluation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblot
Authors: Nomura Sadahiro , Kashiwagi Shiro , Ito Haruhide , Mimura Yusuke , Nakamura Kazuyuki 
Published: May 2005

AbstractTo characterize local hyperfibrinolysis in the pathogenesis of chronic subdural hematoma (CSDH), fibrin degradation products (FDPs) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to identify each FDP fragment (fragments HMW, YY, DY and DD) by immunoblotting. The electrophoretic patterns of FDP in 40 hematomas from 33 patients could be categorized into 3 patterns; (i) type 1, the sharp DY and DD bands; (ii) type 2, the broad DY and DD bands; and (iii) type 3, the sharp DY and DD with two bands with higher molecular weights than fragment DY and DD. The broadening of DY and DD bands was reproduced by incubating the sample containing sharp DD and DY bands with the hemolysed peripheral blood cells. These results indicated that the hyperfibrinolysis in CSDH could be characterized by the constant and incomplete proteolysis of fibrinogen and fibrin by plasmin and further degradation of FDPs by non‐plasmin proteases released from hemolysed blood cells.
Two‐dimensional electrophoresis as an aid in the analysis of the clonality of immunoglobulins
Authors: Tissot Jean‐Daniel , Schneider Philippe , Hohlfeld Patrick , Spertini François , Hochstrasser Denis F. , Duchosal Michel A. 
Published: May 2005

AbstractIn this paper, we summarize our five‐year observation of two‐dimensional polyacrylamide gel electrophoretic (2‐D PAGE) analysis of immunoglobulin (Ig) light (L) chain patterns on serum/plasma and/or purified human Ig, and compare this technique with agarose electrophoresis and/or immunofixation examination. Polyclonal Ig L chains were seen as large “fuzzy” areas with several zones of high density. The majority (71%) of the monoclonal Ig L chains of monoclonal gammopathy detected by conventional electrophoresis appeared as a single large and well‐defined spot on 2‐D PAGE analysis, with the remaining appearing as multiple spots. The presence of oligoclonal Ig, reflected by multiple spots in 2‐D PAGE, and several bands in immunofixation, was observed in 5 of 26 patients after allogeneic bone marrow transplantation and in 5 patients with tumors. In the majority (77%) of hypergamma‐globulinemia, L chains appeared as a wide spread of small and well‐defined spots in 2‐D PAGE analysis. This pattern suggested oligoclonal Ig‐secreting B cell clone expansion, and corresponding abnormalities were not detected with immunofixation. 2‐D PAGE analysis also detected oligoclonal Ig expansions whereas conventional electrophoretic examination was normal in 10 additional patients after bone marrow transplantation, and in 6 of 10 immunocompetent patients with acute severe infections. Analysis of the Ig L chain pattern of a severe combined immune‐deficient mouse populated with human peripheral blood leukocytes confirmed the skewed human Ig production in the model. In summary, 2‐D PAGE appears to be a sensitive tool for the analysis of Ig diversity in various clinical situations.
Colloidal carriers for intravenous drug targeting: Plasma protein adsorption patterns on surface‐modified latex particles evaluated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Blunk Torsten , Hochstrasser Denis F. , Sanchez Jean‐Charles , Müller Bernd W. , Müller Rainer H. 
Published: May 2005

AbstractTargeting to specific sites of the body via colloidal carriers is sought in order to reduce drug side effects. The adsorption of plasma proteins on intravenously injected particles is regarded as the key factor in explaining their organ distribution: total bound protein, or, more likely, the presence of specific proteins and their conformation, are expected to influence macrophage uptake. Polystyrene beads, 60 nm in diameter, were used as model carriers; their surface was differentially modified by adsorption of increasingly hydrophilic block copolymers, poloxamers 184, 188 and 407. After incubation in plasma, the patterns of protein adsorption onto coated beads were analyzed by high‐resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). The behavior of some representative proteins was monitored, including albumin, fibrinogen, IgG, factor B and the apolipoproteins, A‐I, A‐IV, C‐III, E and J. The more hydrophobic the particles, the larger the total amount of bound protein. However, this correlation was not valid for all of the analyzed protein species, which proves that it is insufficient to look only at physico‐chemical data to predict organ distribution. On the contrary, it is essential to use 2‐D PAGE to establish the correlation between adsorbed proteins and carrier behavior in vivo.
Electrophoretic detection of chitinase isoenzymes using the PhastSystem
Authors: McBride Jeffrey D. , Stubberfield Colin R. , Hayes David J. 
Published: May 2005

AbstractZymographic assays are described for the detection of chitinase isoenzymes following isoelectric focusing. Method 1 used a polyacrylamide overlay gel containing glycol chitin. Following hydrolysis, visualisation of isoenzymes was achieved by fluorescent counter‐staining of the gel with Fluorescent Brightener 28. Method 2 used a cellulose acetate membrane overlay which incorporates 4‐methylumbelliferyl substrates, hydrolysis of which released a fluorescent product (4‐methylumbelliferone). The pIs of chitinase isoenzymes were estimated by using coloured pI markers. A significant time advantage was obtained over previous methods. The method was used to demonstrate chitinase activity in control rat lungs and in rat lungs infected with the pathogen Pneumocystis carinii.
Concanavalin A crossed affinoimmunoelectrophoretic analysis of the major pig serum proteins during fetal development
Authors: Lampreave Fermín , Alava María A. , Piñeiro Andrés 
Published: May 2005

AbstractThe interaction between concanavalin A (Con A) and α‐fetoprotein (AFP), transferrin (TF), fetuin, α1‐antitrypsin (AT) and α1‐acid glycoprotein (AGP) has been analyzed by crossed affinoimmunoelectrophoresis (CAIE) in fetal extracts or sera, from 26‐day‐old porcine fetuses to birth, and in adult pigs. Most of the TF and AFP (100 and 85–90%, respectively) reacted with Con A during the entire developmental period. AGP showed both two reactive and one nonreactive Con A isoforms, whose proportions change greatly during development. In younger fetuses 100% of the protein was Con A‐nonreactive. This variant represented 64% in 50‐day‐old fetuses, 80% in newborn pigs and 20% in adult sera. Fetuin and AT showed a maximum of three Con A‐reactive microforms and one Con A‐nonreactive microform, which was always a minor form. These microforms were detected mainly in young fetuses. Although the proportion of Con A‐reactive variants of fetuin and AT changes during fetal development, the predominant microform was always that with intermediate affinity against Con A. The same microform was also predominant in adult AT, whereas the more reactive microform in respect to Con A predominates in adult fetuin.
Capillary electrophoresis of DNA restriction fragments with solutions of entangled polymers
Authors: Kleemiß Maria H. , Gilges Martin , Schomburg Gerhard 
Published: May 2005

AbstractSeparation of DNA restriction fragments in dilute buffer solutions of network forming polymers such as linear polyacrylamide (PAA), hydroxyethyl cellulose (HEC) and polyvinylalcohol (PVA) in phosphate buffer were investigated. PVA in buffers already became inhomogeneous after a few separations with resultant deterioration of resolution. Hydroxylic polymers, capable of forming suitable networks in buffers, are strongly adsorbed to fused silica surfaces suppressing the electroosmotic flow. These polymers could be applied as surface modifiers in capillaries, filled with buffer media containing other polymers such as PAA: Suppression of the electroosmotic flow by adsorbed HEC at pH 7 was slightly more effective than with PVA but the former coating was less stable due to weaker binding to the fused silica surface. Good separations of restriction fragments could be achieved with solutions of PAA or HEC as separation media after surface pretreatment of fused silica capillaries by either PVA rinsing or coating according to Hjertén [41].
Analysis of lipoproteins with analytical capillary isotachophoresis
Authors: Schmitz Gerd , Möllers Christoph 
Published: May 2005

AbstractAn analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well‐characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride‐rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary iso tachophoresis. In addition to patient analysis we investigated the influence of lipid lowering drugs on the lipoprotein subfraction distribution during therapy with analytical capillary isotachophoresis.
Investigation of drug metabolism using capillary electrophoresis with photodiode array detection and online mass spectrometry equipped with an array detector
Authors: Tomlinson Andy J. , Benson Linda M. , Johnson Kenneth L. , Naylor Stephen 
Published: May 2005

AbstractThe application of capillary electrophoresis (CE) with photodiode array detection (DAD) and on‐line CE‐mass spectrometry (CE‐MS) equipped with a position and time resolved (PATRICtm) focal plane detector for analysis of both in vitro and in vivo drug metabolism is demonstrated. Separation of metabolites derived from the neuroleptic drug haloperidol, by CE, using a simple, volatile run buffer containing 50 mM ammonium acetate with 10% methanol and 1% acetic acid is reported. The potential utility of CE‐DAD for screening drug metabolite mixtures derived from hepatic microsomal incubations is demonstrated for haloperidal (HAL). Also the potential problems associated with using this technology to screen human urine samples for HAL metabolites is discussed. Furthermore, the usefulness of CE‐MS and CE‐electrospray ionization skimmer collision induced dissociation‐MS (CE‐ESI‐CID‐MS) in identification and structure elucidation of HAL metabolites derived from both a guinea pig hepatic microsomal incubation and urine from a patient treated with 0.5 mg/day of HAL is shown. The utility of such an approach in the general area of clinical pharmacology is also discussed.
Neutral and anionic cyclodextrins in capillary zone electrophoresis: Enantiomeric separation of ephedrine and related compounds
Authors: Dette Christoph , Ebel Siegfried , Terabe Shigeru 
Published: May 2005

AbstractTwo methods for the enantiomeric separation of chiral ephedrine alkaloids (ephedrine, methylephedrine, methylpseudoephedrine and norephedrine) by capillary zone electrophoresis in uncoated capillaries were developed. Both methods were optimized to more than 100 000 theoretical plate numbers. The first method used a neutral cyclodextrin (CD) derivative: heptakis (2,6–di‐O‐methyl)‐β‐cyclodextrin at an acidic pH of 2.5 (20 mM phosphate buffer) at an 18 mM concentration. The second used a newly developed acidic CD derivative, the tetrakis[6‐O‐(4‐sulfobutyl)]‐β‐cyclodextrin sodium salt. The benefits of this new reagent for the chiral separation are a wide range of basic pH available for the enantiomeric resolution. It is also useful for other cases, e.g. preventing adsorption without additives.
Capillary zone electrophoresis and micellar electrokinetic chromatography of 4‐aminobenzonitrile carbohydrate derivatives
Authors: Schwaiger Hannes , Oefner Peter J. , Huber Christian , Grill Eduard , Bonn Günther K. 
Published: May 2005

AbstractAldoses, ketoses and uronic acids were derivatized successfully within 15 min at a temperature of 90°C by reductive amination with 4‐aminobenzonitrile. Subsequently, the derivatives were separated as their borate complexes by capillary zone electrophoresis, using 175 mM borate buffer, pH 10.5, as carrier. The electrophoretic mobilities were determined by the complex stability, which was found to depend on the number of hydroxyl groups on any given carbohydrate derivative, the presence of substituents, and most strongly on the configuration of the vicinal hydroxyl groups at C‐3 and C‐4 in aldoses and uronic acids, and with regard to ketoses on those at C‐4 and C‐5. Time of analysis could be reduced considerably by the use of micellar electrokinetic chromatography, which separated 4‐aminobenzonitrile sugar derivatives on the basis of their differential partitioning into an electroendosmotically driven aqueous phase and into sodium dodecyl sulfate micelles. Optimum resolution was achieved with a Tris‐phosphate buffer, pH 7.5, containing 100 mM of sodium dedecyl sulfate. The method made it possible to resolve several carbohydrates which had not been resolved successfully by means of capillary zone electrophoresis, such as glucose and fructose. Moreover, separation selectivity could be adjusted by varying the capillary temperature. Finally, on‐column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 1 fmol and a concentration sensitivity of 0.3 μM.
The validity of the Smoluchowski equation in electrophoretic studies of lipid membranes
Authors: Egorova Elena M. 
Published: May 2005

AbstractThe Helmholtz‐Smoluchowski theory, widely used for the calculation of zeta potential from the measured electrophoretic mobility (EPM), is known to be invalid in the region where the mobility is affected by the surface conductivity and polarization of the electrical double layer. In this region, the zeta potential found according to the Smoluchowski equation (ζsm) is not identical to the true electrostatic potential at the hydrodynamic plane of shear (ζ) which is considered in the Gouy‐Chapman‐Stern theory of the electrical double layer. As a result, ζsm cannot be used for the subsequent calculation of surface potential and surface charge density of a membrane studied. Here we suggest a simple way, allowing one to decide between the validity and nonvalidity of the Smoluchowski equation in various sets of experimental conditions used in electrophoretic measurements on lipid membranes. We calculated the dimensionless criterion Rel, accepted in the Dukhin theory of electrophoresis as a measure of the extent of the influence of surface conductivity and double layer polarization on EPM. The Rel changes, with membrane charge density, ionic strength and vesicle radius, were found to be helpful in finding the combinations of these three parameters, corresponding to the validity of the Smoluchowski equation.
Polypeptide cartography of Spiroplasma taiwanense
Authors: Humphery‐Smith Ian , Guyonnet Franck , Chastel Claude 
Published: May 2005

AbstractSpiroplasma taiwanense is the first member of the Class Mollicutes to be subjected to polypeptide cartography using computerized image analysis. The small genome size characteristic of this group was shown to code for low numbers of polypeptides when compared to other bacterial species. Silver‐stained two‐dimensional electrophoresis gels, following separation by either isoelectric focusing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (ISO‐DALT) or nonequilibrium pH gradient electrophoresis (NEPHGE), were used to create databases from 10 and 6 gels, respectively, for each technique and produced, respectively, 263 and 287 replicated spots. Polypeptides were mapped with respect to molecular mass and glyceraldehyde‐3‐phosphate dehydrogenase carbamylation standards. Of interest was the unexpectedly high percentage (50.2%) of the total normalised optical intensity associated with all 263 spots detected by ISO‐DALT electrophoresis, having been contributed by just 29 dominant protein spots. These 29 polypeptides are to be given priority in microsequencing and microanalysis aimed at their identification.