bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isoelectric focusing and non‐isoelectric precipitation of ferritin in immobilized pH gradients: An improved protocol overcoming protein‐matrix interactions
Authors: Righetti Pier Giorgio , Gelfi Cecilia , Bossi Maria Luisa , Boschetti Egisto 
Published: May 2005

AbstractIt has been impossible, up to now, to focus ferritin (a large, ca. 450 000 Da, ironstorage protein ubiquitous in mammalian systems) in immobilized pH gradients (IPG). This was not a sieving problem, as ferritin focuses readily in a 4%T polyacrylamide (or polytrisacryl) gel in conventional carrier ampholyte isoelectric focusing. A strong interaction with the IPG matrix was suspected, but an auto‐buffered (pH 6.0) soluble IPG polymer failed to precipitate ferritin from solution, although it altered its visible spectrum, suggesting the formation of soluble complexes. By a Takeo plot, the dissociation constant (Kd)of the ferritin‐IPG matrix complex was estimated to be 0.1 mM. However, even a 100‐fold dilution of Immobilines into the IPG matrix with a concomitant addition of competing ions (up to 3 % carrier ampholytes) failed to produce good focusing patterns. An unsuspected, hitherto un‐reported, precipitating power of free Immobilines in solution on the ferritin macroion was found to be the key to the riddle. The precipitating ions are (in order of aggregation power): pK 9.3 > pK 7.0 > pK 6.2. None of the acidic Immobilines (pKs 3.6, 4.4 and 4.6) and, among the basic, only the pK 8.5, exhibit any precipitation power on ferritin and other complex protein mixtures. In a new IPG pH 4–5 interval, containing only the pK 4.6 as buffering and the pK 8.5 Immobilines as titrant ions, excellent focusing patterns of ferritin were obtained. It is hypothesized that the precipitating Immobilines act as cationic detergents, and are able to cross‐link macroions with a mixed‐type, hydrophobic‐ionic interaction at the two extremities of the molecule. Guidelines are given for handling this major problem on a short‐term basis, the longterm goal being the replacement of the ‘unfavorable’ Immobilines (pKs 9.3, 7.0 and 6.2) by new chemical structures of the desirable type (the non‐precipitating pK 8.5 species).
Cyanogen bromide‐activated nitrocellulose membranes: A new tool for immunoprint techniques
Authors: Demeulemester Claude , Peltre Gabriel , Laurent Micheline , Panheleux Dominique , David Bernard 
Published: May 2005

AbstractNitrocellulose membranes were activated by cyanogen bromide in order to improve their binding capacities. Crude extracts of a grass pollen, Dactylis glomerata, and a mite, Dermatophagoides farinae, were separated by isoelectric focusing in agarose and blotted by capillary transfer onto nitrocellulose or activated nitrocellulose. The Dermatophagoides farinae components were visualized by Coomassie Brilliant Blue, India ink or Aurodye staining. The Dactylis glomerata and Dermatophagoides farinae allergens were revealed by radioimmunodetection using sera from allergic patients. A great increase of allergen binding was observed visually and by radioactive counting on activated nitrocellulose as compared with untreated nitrocellulose. New allergens which were not detectable on nitrocellulose appeared on activated nitrocellulose.
Studies on the heterogeneity of anti‐hapten antibodies by means of two‐dimensional affinity electrophoresis
Authors: Takeo Kazusuke , Tanaka Tatehiko , Nakamura Kazuyuki , Suzuno Ryosuke 
Published: May 2005

AbstractThe molecular heterogeneity of rabbit anti‐hapten antibodies has been investigated by two‐dimensional affinity electrophoresis (2D‐AEP). Anti‐dansyl and anti‐arsanilic diazo‐antibodies were separated into several hundred IgG spots as in the case of anti‐DNP antibodies. They were grouped into a number of monoclonal IgG families. At the beginning of immunization, IgG spots having low pI and low affinity were predominant but one or two weeks after immunization the IgG spots with high affinity and high pI increased. After the second or third immunization, the 2D‐AEP patterns became stable and constant. Anti‐arsanilic diazo and anti‐DNP antibodies exhibited only weak cross‐reactivity with other aromatic haptens. In contrast, anti‐dansyl antibodies cross‐reacted to a considerable degree with DNP‐hapten. A few anti‐dansyl IgG families which have cross‐reactivity with DNP‐hapten were separated. Their apparent dissociation constants to the haptens and their affinity ratios were calculated from their 2D‐AEP patterns, according to the affinity theory.
Concanavalin A crossed affinity immunoelectrophoresis and image analysis for semiquantitative evaluation of microheterogeneity profiles of human serum transferrin from alcoholics and normal individuals
Authors: Heegaard Niels H. H. , Hagerup Martin , Thomsen Åge C. , Heegaard Peter M. H. 
Published: May 2005

AbstractThe microheterogeneity profile of human serum transferrin from normal and alcoholic subjects was investigated qualitatively and quantitatively by means of Concanavalin A crossed affinity immunoelectrophoresis and an image analysis program. Differences in amounts of nonreacting transferrin molecules were found, suggesting an increase in triantennary glycosylation of transferrin from alcoholics compared with normal individuals. The increased amount of a highly retarded fraction in crude sera from alcoholics was demonstrated to be artefactual, probably due to entrapment or coprecipitation as the fraction disappeared after repeating the analysis with immunosorbent‐purified transferrin. In conclusion, affinity electrophoresis represents a simple approach for demonstration of variations in the neutral monosaccharides of glycans and can discriminate between transferrin from alcoholics and normal individuals.
Affinophoresis of red blood cells: Surface antigen‐specific acceleration of electrophoresis
Authors: Shimura Kiyohito , Ogasawara Norikazu , Kasai Ken‐ichi 
Published: May 2005

AbstractA method employing the technique of affinophoresis to increase the electrophoretic mobility of specific cells according to their surface antigens was developed. Red blood cells were treated consecutively with the maximum subagglutinating dose of an antired blood cell serum, a biotinylated second antibody, avidin and finally with a negatively charged biotin‐affinophore which was prepared by coupling biotin to polylysine (average degree of polymerization, 270 or 1150), followed by complete succinylation. The electrophoretic mobility of cells was analyzed with an automatic cell electrophoresis analyzer. The use of a homologous anti‐serum increased the electrophoretic mobility of rabbit, human and rat red blood cells by 2.9, 1.7 and 1.6 times, respectively. A larger affinophore containing fewer biotin moieties was more effective. In the case of a mixture of red blood cells from two species, cells from only one species could be accelerated by using homologous antiserum, e.g., affinophoresis of a mixture of human and rat red blood cells by using either homologous antiserum gave two separate peaks on the histogram, whereas a single peak would be obtained in usual electrophoresis because there is little difference in the original migration velocities of the two cell types.
DNA sequencing in HydroLink matrices: Extension of reading ability to > 600 nucleotides
Authors: Gelfi Cecilia , Canali Alessandra , Righetti Pier Ciorgio , Vezzoni Paolo , Smith Carolyn , Mellon Mark , Jain Tikam , Shorr Robert 
Published: May 2005

AbstractAll the systems for optimizing DNA sequencing published so far have introduced modifications regarding: (i) linearization of band migration via ionic strength gradients or wedge‐shaped gels; (ii) automatization of band reading via introduction of fluorescent probes; (iii) direct blotting analysis; (iv) pulsed electric fields and (v) discontinuous buffer systems. In all these systems, DNA sequence reading with an accuracy of ca. 98 % rarely exceeds a length of 350 bases. We have chosen, in order to increase the reading ability of a single gel, to manipulate the characteristics of the gel matrix. The Seq‐HydroLink gel formulation here reported allows optimal reading, from a single gel run, of at least 600 bases. In order to guarantee this reading ability in a single run, the upper and lower ends of the ladder are time‐resolved, i. e. the same sample is applied to the gel matrix at three different time intervals. The present system represents an increase of at least 30 % in reading ability as compared with any type of polyacrylamide gel formulation so far reported.
Isoproteins and an isoelectric focusing mutant of human apoprotein serum amyloid A
Authors: Steinmetz Armin , Schmidt Barbara , Hocke Gerti , Motzny Sabine , Vitt Herbert , Kaffarnik Hans 
Published: May 2005

AbstractOn isoelectric focusing of human plasma and subsequent immunoblotting, using antii‐human serum amyloid A (SAA) antibodies, a genetic variant of SAA was detected in a family of Turkish origin. All affected members of the family were apparent heterozygotes or the mutant protein, which underwent a charge shift of about one charge unit toward the anode. The variant is likely to be a mutant of the most prominent forms of SAA (SAA1 and SAA2, or SAA1 and SAA1 des Arg). The appearance of a genetic variant of two of the six reported SAA‐isoforms in human plasma supports the concept of SAA proteins being products of different genes.
Direct immunoblotting from histological sections of brain onto nitrocellulose: Evaluation with the protein neurophysin
Authors: Reisinger Peter W. M. , Unger Jürgen W. 
Published: May 2005

AbstractA method for the isolation and localization of proteins and peptides from histological sections of rat and human brain by immunoblottin is described. For validation, the well‐characterized protein neurophysin was electrophoretically transferred from formaldehyde‐fixed or fresh tissue sections onto a nitrocellulose membrane. Neurophysin on the nitrocellulose membrane was detected by a specific antibody reaction. The antibody against neurophysin was visualized either by using secondary antibodies, conjugated with peroxidase or by protein A gold, followed by enhancement with silver. With this simple and fast method, neurophysin (or other proteins and peptides) can be identified on nitrocellulose membranes in areas that correspond to anatomically defined regions. Since the procedure combines the advantages of precise regional localization of polypeptides with the specificity of antibody‐antigen reactions, the method may prove useful for rapid screening of the distribution of peptides or proteins in (brain) tissue.
Involvement of cysteine residues in the electrophoretic mobility of histone H3 in acid‐urea‐Triton gels
Authors: Waterborg Jakob H. 
Published: May 2005

AbstractCarbamylation of cysteines 96 and 110 in histone H3 increases the electrophoretic mobility of this histone in acetic acid‐urea‐Triton X‐100 polyacrylamide gels but has no effect in gels lacking Triton. Residue 96 appears to be a major determinant in the affinity of histone H3 for the nonionic detergent Triton. Carbamylation and carboxymethylation of cysteine 96 caused a major loss of the gel retardation caused by Triton. Carbamylation of cysteine 110 did not affect Triton binding but prevented ionization of the thiol side‐chain moiety in the acetic acid‐urea‐Triton X‐100 gel.
Serum proteins of rats exposed to organic solvents examined by horizontal two‐dimensional electrophoresis with an immobilized pH gradient in the first dimension
Authors: Zastrow Gabriela , Günther Siegfried , Görg Angelika , Postel Wilhelm , Diehl Horst A. , Jansen Eugéne H. J. M. 
Published: May 2005

AbstractSerum proteins of exposed to methoxyethylacetate and a combination fo methoxyethylacetate and isobutylacetate, were analyzed horizontal high resolution two‐dimensional gel electrophoresis with immobilized pH gradienets in the first dimension. A total of 410 polypeptide were detected with either increasing or decreasing spot intensities after rat exposure to organic solvents.
Tumor suppressor p53 gene forms multiple isoforms: Evidence for single lucus origin and cytoplasmic complex formation with heat shock proteins
Authors: Selkirk James K. , He Chaoying , Patterson Rachel M. , Merrick B. Alex 
Published: May 2005

AbstractThe tumor suppressor protein p53 is a major cell cycle control factor, and mutations in p53 are the most common genetic lesion found in human tumors, resulting in loss of function and contributing to malignant transformation. This report reviews several studies which show that p53 protein appears as at least eleven isoforms having the same amino acid backbone but varying in charge by level of phosphorylation. All isoforms are derived from a single locus, which indicates that p53 activity is modulated by post‐translational modification. In addition, mutant p53 forms hetero‐oligomers with two families of proteins: HSP70 and a 90 kDa group similar to HSP90. Cytoplasmic complexes are most likely formed to protect p53 from proteolysis and are probably involved in translocation of activated p53 from the cytoplasm to the nucleus for transactivation of other cell cycle control genes.
Analysis of phosphorylated isoforms of the p53 tumor suppressor protein in human lung carcinoma cells undergoing apoptosis
Authors: Maxwell Steve A. , Roth Jack A. , Mukhopadhyay Tapas 
Published: May 2005

AbstractThe expression of p53 tumor suppressor protein isoforms in H460a cells induced to undergo apoptosis by 2‐methoxyestradiol was investigated by two‐dimensional gel electrophoresis. Whole‐cell proteins from control and apoptotic H460a cells were separated by two‐dimensional electrophoresis and were transferred to nitrocellulose. The p53 isoforms were detected by immunoblotting using p53 monoclonal antibody Bp53‐12. Four isoforms of p53 (2, 3, 5, and 6) differing in phosphorylation state were detected in control cells. Three additional isoforms (1, 4, and 7) were observed to be expressed at significant levels only in apoptotic cells. The differential expression of isoforms 1, 4, and 7 in apoptotic cells suggests that one or more specific phosphorylation events generating these forms of p53 could play a role in regulating the function of p53 in apoptosis.
Use of two‐dimensional gel electrophoresis to characterize protein synthesis during neuronal death in cerebellar culture
Authors: Charriaut‐Marlangue Christiane , Dessi Frédéric , Ben‐Ari Yezekiel 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis was performed to further investigate the biochemical changes in protein synthesis observed in two neuronal death models, induced respectively by cytosine arabinoside and glutamate. These drugs induced, respectively, apoptotic and necrotic types of cell death in cerebellar cultures, as previously reported. Most of the proteins showed decreased labeling after toxic exposure, as expected, but some polypeptides showed increased labeling or appeared to be newly synthesized. The identification of these polypeptides and their implication in neuronal death are discussed.
A critical evaluation of the application of capillary electrophoresis to the detection and determination of 1,4‐benzodiazepine tranquilizers in formulations and body materials
Authors: Smyth W. Franklin , McClean Stephen 
Published: May 2005

AbstractStudies of the capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) behaviour of 1,4‐benzodiazepines have seen application in subject areas such as the development of pharmaceuticals, therapeutic drug monitoring and forensic toxicology. In the development of pharmaceuticals, pKa determinations by CZE can be used in preclinical studies whereas analytical data on the detection and determination of 1,4‐benzodiazepines is of value primarily in raw material/formulation assay and in the analysis of body fluids in clinical studies. The capillary electrophoresis (CE) techniques, which generally have inferior limits of detection (LOD) to rival techniques such as gas chromatography (GC) and high‐performance liquid chromatography (HPLC), are particularly applicable in forensic toxicology where reasonably high concentrations of these drugs can be encountered. It is anticipated that, with the interfacing of CZE and capillary electrochromatography (CEC) with mass spectrometry (MS) techniques, the excellent selectivity of CZE and particularly CEC will be effectively combined with the sensitivity of MS and the identification capabilities of tandem mass spectrometry (MS/MS) and MS hyphenated (MSn) techniques.
Selectivity manipulation using nonaqueous capillary electrophoresis. Application to tropane alkaloids and amphetamine derivatives
Authors: Cherkaoui Samir , Varesio Emmanuel , Christen Philippe , Veuthey Jean‐Luc 
Published: May 2005

AbstractNon‐aqueous capillary electrophoresis was investigated for its potential in the analysis of pharmaceutical compounds, namely tropane alkaloids and amphetamine derivatives. The separation of these drugs was compared in aqueous and organic media such as methanol and/or acetonitrile. Selectivity, migration times and efficiency were critically affected by the composition of the methanol/acetonitrile mixture, as well as by the nature and the concentration of the electrolyte. In particular, the migration orders of two positional isomers, littorine and hyoscyamine, were inverted in the presence of trifluoroacetic acid in the nonaqueous medium. The same behavior was observed for amphetamine‐methamphetamine and for two methylenedioxyamphetamine derivatives.
Two examples of rapid and simple drug analysis in pharmaceutical formulations using capillary electrophoresis: Naphazoline, dexamethasone and benzalkonium in nose drops and nystatin in an oily suspension
Authors: Raith Klaus , Althoff Eva , Banse Jutta , Neidhardt Hiltrud , Neubert Reinhard H. H. 
Published: May 2005

AbstractCapillary electrophoresis is a versatile tool in pharmaceutical analysis. In the course of a revision of the “Standardrezepturen”, a German formula of standard dispensings for preparation in pharmacies, this technique has been applied to drug analysis in pharmaceutical formulations. The present paper deals with two different examples. First, naphazoline, dexamethasone and the preservative benzalkonium are quantified in nose drops without any sample preparation. Second, the antifungal antibiotic nystatin is quantified using nonaqueous capillary electrophoresis in methanol after sample preparation from an oily suspension.
Screening for urinary amphetamine and analogs by capillary electrophoretic immunoassays and confirmation by capillary electrophoresis with on‐column multiwave‐length absorbance detection
Authors: Ramseier Andreas , Caslavska Jitka , Thormann Wolfgang 
Published: May 2005

AbstractThis paper characterizes competitive binding, electrokinetic capillary‐based immunoassays for screening of urinary amphetamine (A) and analogs using reagents which were commercialized for a fluorescence polarization immunoassay (FPIA). After incubation of 25 μL urine with the reactants, a small aliquot of the mixture is applied onto a fused‐silica capillary and unbound fluorescein‐labeled tracer compounds are monitored by capillary electrophoresis with on‐column laser‐induced fluorescence detection. Configurations in presence and absence of micelles were investigated and found to be capable of recognizing urinary D‐(+)‐amphetamine at concentrations > about 80 ng/mL. Similar responses were obtained for racemic methamphetamine (MA) and 3,4‐methylenedioxymethamphetamine (MDMA). The electrokinetic immunoassay data suggest that the FPIA reagent kit includes two immunoassay systems (two antibodies and two tracer molecules), one that recognizes MA and MDMA, and one that is geared towards monitoring of A. For confirmation analysis of urinary amphetamines and ephedrines, capillary electrophoresis in a pH 9.2 buffer and multiwavelength UV detection was employed. The suitability of the electrokinetic methods for screening and confirmation is demonstrated via analysis of patient and external quality control urines.
Analysis of fluorescein isothiocyanate derivatized amphetamine and analogs in human urine by capillary electrophoresis in chip‐based and fused‐silica capillary instrumentation
Authors: Ramseier Andreas , Heeren Franz Von , Thormann Wolfgang 
Published: May 2005

AbstractAmines can easily be derivatized with fluorescein isothiocyanate isomer I (FITC) and analyzed by capillary electrophoresis (CE) using alkaline buffers with or without dodecyl sulfate micelles. This paper reports the CE analysis of FITC‐derivatized amphetamine, methamphetamine, 3,4‐methylenedioxymethamphet‐amine and β‐phenylethylamine in human urine using chip‐based and fused‐silica capillary instrumentation with laser‐induced fluorescence detection. Data obtained via direct labeling of fortified urine are compared to those generated after FITC labeling of urinary extracts that were prepared by solid‐phase extraction using a copolymer phase. For a urine volume of 5 mL with a “spiked amine”: FITC ratio of 1:250, the latter approach was found to provide a sensitivity that is relevant for toxicological drug screening and confirmation (about 200 ng/mL urine). With direct labeling of 10 μL urine that was alkalinized and diluted for derivatization, the limit of identification was determined to be about 10 μg/mL, a value that is too high for practical purposes. Compared to fused‐silica capillaries, electrophoresis in microstructures is shown to provide faster separations and higher efficiencies without loss of accuracy and precision.
Serum procainamide analysis based on acetonitrile stacking by capillary electrophoresis
Authors: Shihabi Zakariya K. 
Published: May 2005

AbstractStacking methods are important in capillary electrophoresis (CE) to overcome the poor detection limits. Cationic drugs are difficult to stack because they tend to interact with the capillary wall. As an example of the stacking of the cationic compounds, procainamide, an anti‐arrhythmic drug, is analyzed in serum by CE using an acetonitrile treatment. Serum was deproteinized with acetonitrile containing quinine as an internal standard. About 12% of the capillary volume was filled with sample and separated using an electrophoresis buffer composed of triethanolamine, 2‐(N‐cyclohexylamino)ethanesulfonic acid (CHES) and 20% isopropanol, pH 8.2. Both the triethanolamine and the CHES were critical for the stacking. The addition of isopropanol improved the plate number for the procainamide and decreased the interfering compounds. Procainamide, its metabolite N‐acetyl procainamide, and quinine were separated in about 7 min. The CE compared well with an immunoassay method.
Capillary ion analysis of lithium concentrations in biological fluids and tissues of Poecilia (teleost)
Authors: Creson Thomas K. , Monaco Paul J. , Rasch Ellen M. , Hagardorn Andrea H. , Ferslew Kenneth E. 
Published: May 2005

AbstractCapillary ion analysis (CIA) is a form of capillary electrophoresis that uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Application of this technique for detection of lithium concentrations in plasma and tissues of Poecilia was the purpose of this investigation. CIA was performed using a 75 μm ID × 60 cm length fused‐silica capillary and a run electrolyte of 67.7 mg hydroxyisobutyric acid (HIBA), 52.8 mg 18‐crown‐6‐ether and 64 μL UV‐CAT‐1 reagent (4‐methylbenzylamine) in a volume of 100 mL water (18 MΩ) with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: potassium, 2.98 min; calcium, 3.48 min; sodium, 3.60 min; barium (internal standard), 4.15 min and lithium, 4.26 min. Lithium and barium migration times were stable and reproducible. Correlation coefficients (r) between peak area ratios of lithium/barium for concentrations ranging from 0.1 to 2.0 mM were from 0.976 to 0.996. Coefficients of variation (CV) for lithium concentrations ranged from 4.07 to 15.71% between days and 4.38 to 7.76% within‐day. Application of this methodology for determination of lithium concentrations in the plasma, brains and livers of fish dosed with lithium for 23 days are presented. CIA is applicable to analysis of lithium concentrations in biological fluids and tissues of fish.