bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analytical capillary isotachophoresis of human serum lipoproteins
Authors: Schmitz Gerd , Möllers Christoph , Richter Volker 
Published: May 2005

AbstractAn analytical free flow capillary isotachophoresis (cITP) procedure for the detailed analysis of lipoproteins on commercially available capillary electrophoresis systems has been developed. The technique is based on the specific staining of lipoproteins with the fluorescent lipophilic dye 7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD)‐ceramide before separation. Prestained lipoprotein samples are applied between leading and terminating buffer and separated into 9 well‐characterized subpopulations according to their electrophoretic mobility in the absence of any molecular sieve effect. High density lipoproteins are separated into three major subpopulations: (i) the fast migrating high density lipoprotein (HDL) subpopulation (α‐HDL, containing mainly apolipoprotein (apo) A‐I and phosphatidylcholine, (ii) the subpopulation with intermediate mobility, consisting of particles rich in cholesterol, apo A‐II, apo E and C apolipoproteins, and (iii) the slow migrating HDL subpopulation (pre‐β‐HDL), containing particles rich in apo A‐I, apo A‐IV. The majority of HDL‐associated lecithin:cholesterol acyltransferase (LCAT) activity is also associated with the last subpopulation. The apo B‐containing lipoproteins can be subdivided into three 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, and the third group represents the low density lipoproteins (LDL). Results obtained by the isotachophoretic lipoprotein analysis revealed a good correlation in the range of HDL with routinely used techniques, like lipoprotein electrophoresis, HDL‐cholesterol analysis by a precipitation procedure or turbidi‐metric determination of apo A‐I. Similar correlations with other analytical techniques were found for the quantitation of the apo B‐containing lipoproteins. Advantages of the isotachophoretic separation compared to zone electrophoresis are the high resolution combined with small sample volumes. Moreover, lipoprotein analysis can be performed directly from whole serum, plasma, lymph and other biological fluids in a short time. With these characteristics analytical capillary isotachophoresis may be a helpful tool for a fast and reliable automated quantitation of lipoprotein subpopulations in the clinical laboratory.
Urine protein analysis by capillary electrophoresis
Authors: Friedberg Michael A. , Shihabi Zakariya K. 
Published: May 2005

AbstractIncreased concentration of proteins in urine as well as abnormal patterns are seen in many disorders such as various renal disorders and light chain disease. At the wavelength of 214 nm used for detection of the peptide bond, numerous compounds interfere in the analysis of urinary proteins. We show that either adsorptive filtration with a wash step or cold ethanol precipitation are two methods which can eliminate many of the interferences. The wash step is rapid, greatly reduces the interfering substances, and decreases the effect of sample matrix. Both of these methods yield results comparable to the traditional agarose method. Capillary electrophoresis (CE) is faster and more cost‐effective than agarose electrophoresis.
Clinical application of capillary electrophoresis to unconcentrated human urine proteins
Authors: Jenkins Margaret A. 
Published: May 2005

AbstractUrine protein electrophoresis can be performed by capillary electrophoresis using spun unconcentrated urine diluted with running buffer. The separation which utilises the excellent sensitivity of capillary electrophoresis gives electropherograms similar to those obtained by diluting concentrated urine with buffer. A 72 cm × 50 μm internal diameter (ID) fused silica capillary was used for the analysis which took less than 15 min. Bence Jones protein can be detected from unconcentrated urine over urine protein concentrations ranging from 9.7 g/L to 0.04 g/L. Other clinical patterns, such as glomerular protein‐uria, the presence of intact immunoglobulin and Tamm Horsfall protein can also be detected. The benefit of using unconcentrated urine is the time and cost saved by not concentrating the urine.
Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels
Authors: Zerbetto Elisabetta , Vergani Lodovica , Dabbeni‐Sala Federica 
Published: May 2005

AbstractBlue native‐polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi‐subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin‐responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.
Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: Theory and methodology
Authors: Righetti Pier Giorgio , Gelfi Cecilia , Perego Marilena , Stoyanov Alexandre V. , Bossi Allessandra 
Published: May 2005

AbstractThe use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 μm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion‐driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (β) as a function of ΔpK. i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative β value (= 2) is obtained for amphoteres possessing a ΔpK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the β/λ ratio, i.e. the ratio between the β power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of ΔpK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human β‐globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.
Affinity capillary electrophoresis
Authors: Rippel Geza , Corstjens Hugo , Billiet Hugo A. H. , Frank Johannes 
Published: May 2005

AbstractThe application of affinity capillary electrophoresis (ACE) to the study of molecular interactions is reviewed. ACE appears to be a sensitive, versatile and convenient tool to obtain reliable data on binding constants and stoichiometries of interacting systems using the Hummel‐Dreyer method and variants thereof. A powerful feature is the possibility to analyze simultaneously the affinity of a large number of compounds for the same ligand, making it a promising tool for the screening of large combinatorial libraries.
Methods for the estimation of binding constants by capillary electrophoresis
Authors: Rundlett Kimber L. , Armstrong Daniel W. 
Published: May 2005

AbstractCapillary electrophoresis (CE) has developed into a particularly effective means to determine apparent equilibrium constants for molecular association in solution (e.g., to micelles, cyclodextrins, antibiotics, proteins, RNA, DNA. etc.). The various experimental, graphical and mathematical approaches for determining association constants are reviewed. In CE, association constants can be calculated because there is a relationship between substrate concentration and the measured electrophoretic mobility of the solute. Most of the approaches for obtaining association constants by CE are conceptually and mathematically related to one another. Likewise, they are analogous to many spectroscopic techniques that are used for obtaining association constants. The advantages, limitations and proper use of the various CE approaches are examined.
Integrated chip‐based capillary electrophoresis
Authors: Effenhauser Carlo S. , Bruin Gerard J. M. , Paulus Aran 
Published: May 2005

AbstractIntegrated capillary electrophoresis (ICE) is emerging as a new analytical tool allowing fast, automated, miniaturized and multiplexed assays, thus meeting the needs of the pharmaceutical industry in drug development. The current state‐of‐the‐art of ICE is described with an emphasis on the choice of the support material (glass or polymeric materials), electrokinetic fluid handling, and injection and detection issues. Strategies and chip designs for pre‐ or post‐column derivatization, DNA sequencing, on‐line PCR analysis, on‐chip enzymatic sample digestion, fraction isolation, and immunoassays are presented. The review concludes with a brief outlook.
Capillary electrophoresis in clinical and forensic analysis
Authors: von Heeren Franz , Thormann Wolfgang 
Published: May 2005

AbstractDuring the past decade, capillary electrophoresis (CE) emerged as a promising, effective and economic approach for separation of a large variety of substances, including those encountered in clinical and forensic analysis. Reliable and automated CE instruments became commercially available and promoted the exploration of an increasing number of CE methods and fields of application. The widespread applicability of CE, its enormous separation power and high‐sensitivity detection schemes make this technology an attractive and promising tool. This review discusses the principles and important aspects of CE‐based assays and provides an overview of the key achievements encountered with CE in clinical and forensic analysis, including those associated with the analysis of serum proteins, hemoglobin variants, drugs and nucleic acids. Validated assays, interesting applications and future trends in clinical and forensic analysis are also discussed.
Receptor‐mediated endocytosis in kidney proximal tubules: Recent advances and hypothesis
Authors: Marshansky Vladimir , Bourgoin Sylvain , Londoño Irene , Bendayan Moïse , Maranda Bruno , Vinay Patrick 
Published: May 2005

AbstractPreparation of kidney proximal tubules in suspension allows the study of receptor‐mediated endocytosis, protein reabsorption, and traffic of endosomal vesicles. The study of tubular protein transportin vitrocoupled with that of the function of endosomal preparation offers a unique opportunity to investigate a receptor‐mediated endocytosis pathway under physiological and pathological conditions. We assume that receptor‐mediated endocytosis of albumin in kidney proximal tubulesin situandin vitrocan be regulated, on the one hand, by the components of the acidification machinery (V‐type H+‐ATPase, Cl−‐channel and Na+/H+‐exchanger), giving rise to formation and dissipation of a proton gradient in endosomal vesicles, and, on the other hand, by small GTPases of the ADP‐ribosylation factor (Arf)‐family. In this paper we thus analyze the recent advances of the studies of cellular and molecular mechanisms underlying the identification, localization, and function of the acidification machinery (V‐type H+‐ATPase, Cl−‐channel) as well as Arf‐family small GTPases and phospholipase D in the endocytotic pathway of kidney proximal tubules. Also, we explore the possible functional interaction between the acidification machinery and Arf‐family small GTPases. Finally, we propose the hypothesis of the regulation of translocation of Arf‐family small GTPases by an endosomal acidification process and its role during receptor‐mediated endocytosis in kidney proximal tubules. The results of this study will not only enhance our understanding of the receptor‐mediated endocytosis pathway in kidney proximal tubules under physiological conditions but will also have important implications with respect to the functional consequences under some pathological circumstances. Furthermore, it may suggest novel targets and approaches in the prevention and treatment of various diseases (cystic fibrosis, Dent's disease, diabetes and autosomal dominant polycystic kidney disease).
Protein expression profiles in human breast ductal carcinoma and histologically normal tissue
Authors: Bini Luca , Magi Barbara , Marzocchi Barbara , Arcuri Felice , Tripodi Sergio , Cintorino Marcella , Sanchez Jean‐Charles , Frutiger SÉVerine , Hughes Graham , Pallini Vitaliano , Hochstrasser Denis F. , Tosi Piero 
Published: May 2005

AbstractReference two‐dimensional (2‐D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty‐five spots have been assigned by gel matching to the human liver SWISS‐2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N‐terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl‐prolyl cis‐trans isomerase, collagen‐binding protein 2, fructose bisphosphate aldolase, glyceraldehyde‐3‐phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin β isoform and macrophage migration inhibitory factor (MIF). The cancer‐ and tissue‐specificity of the described pattern was assessed by matching to the Swiss‐2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.
Improved detection of P53 mutations in soft tissue tumors using new gel composition for automated nonradioactive analysis of single‐strand conformation polymorphism
Authors: Schneider‐Stock Regine , Haeckel Carsten , Radig Kathrin , Pein Claus‐Dietmar , Roessner Albert 
Published: May 2005

AbstractWe report a new nonradioactive method to detect sequence changes, including single‐base substitutions through shifts in electrophoretic mobility using an automated fluorescence sequencer (ALFexpress, Pharmacia, Biotech) connected to external cooling equipment. Single strands were identified by incorporation of fluorescein‐labeled primers during amplification and subsequent laser detection at the bottom of the gel. The amplified polymerase chain reaction (PCR) products were heat‐denatured and loaded onto a polyacrylamide gel under nondenaturing conditions and strict control of constant low temperature. Peak shifts in the fluorogram indicated mutations. A novel gel composition improved the detection rate for mutations considerably. Automatic analysis of single‐strand conformation polymorphism (SSCP) gels saves time and costs, and is highly reproducible. The method was applied for mutation screening in exon 7 of the p53 tumor suppressor gene in DNA of freshly frozen soft tissue tumors. The mutation spectrum and frequency in exon 7 of the p53 gene are discussed with respect to oncogenesis in soft tissue sarcomas.
Peptides adsorbed on reverse‐phase chromatographic beads as targets for femtomole sequencing by post‐source decay matrix assisted laser desorption ionization‐reflectron time of flight mass spectrometry (MALDI‐RETOF‐MS)
Authors: Gevaert Kris , Demol Hans , Puype Magda , Broekaert Daniël , De Boeck Stefaan , Houthaeve Tony , Vandekerckhove Joël 
Published: May 2005

AbstractWe here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse‐phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization‐reflectron time of flight (MALDI‐RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide‐matrix mixtures. This permits high yield identification and sequence tagging by post‐source‐decay analysis on peptides derived from proteins only available in the femtomole range from one‐dimensional (1‐D) or two‐dimensional (2‐D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.
Large‐scale identification of proteins of Haemophilus influenzae by amino acid composition analysis
Authors: Fountoulakis Michael , Juranville Jean‐François , Berndt Peter 
Published: May 2005

AbstractTwo‐dimensional protein maps of microorganisms are useful tools for elucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. We applied amino acid composition analysis, following separation by two‐dimensional gel electrophoresis, for large‐scale identification of proteins of Haemophilus influenzae. H. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading frames, has been sequenced. For amino acid analysis, we used both precolumn derivatization of amino acids followed by reversed‐phase chromatography of the derivatized residues and post‐column derivatization of the residues previously separated on an ion exchanger. The composition analyses derived from both methods allowed the identification of 110 protein spots. The proteins were identified using the AACompIdent software on the ExPASy server accessible via the World Wide Web with a success rate of 52%. In some cases, introduction of the analysis data of 12 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambiguously identified. Amino acid composition analysis proved to be an error‐robust, efficient method for protein identification. The method can be practically established in every biochemical laboratory and, complementary to mass spectrometry, represents an important analytical tool for the mapping of the proteomes of organisms of interest.
Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection
Authors: Isenberg Alice R. , Allen Ralph O. , Keys Kathleen M. , Smerick Jill B. , Budowle Bruce , McCord Bruce R. 
Published: May 2005

AbstractA series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60°C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clarlyPresented in part at the 7th Annual Frederick Conference on Capillary Electrophoresis. Frederick, MD, USA, October 21–23, 1996.
Theoretical studies on the mobility‐shift assay of protein‐DNA complexes
Authors: Cann John R. 
Published: May 2005

AbstractThe theory of mass transport coupled to revesible macromolecular interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility‐shift behavior of protein‐DNA complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putative cage effect; (iii) cooperative binding to multiple sites; (iv) formation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooperative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target genes. Favorable comparison of simulated with experimental mobility‐shift behavior indicates that the phenomenological mechanisms, whereby observed mobility‐shift patterns are generated during electrophoresis, are embodies in the theory. These studies have provided guidelines for definitive interpretation of mobility‐shift assays and for the design of experiments to develop a detailed understanding of the particular system under investigation.
Activity staining on polyacrylamide gels of trypsin inhibitors from leaves of sweet potato (Ipomoea batatas L. Lam) varieties
Authors: Hou Wen‐Chi , Lin Yaw‐Huei 
Published: May 2005

AbstractThe failure of activity staining of trypsin inhibitors in crude leaf extracts of sweet potatoe varieties including Tainong 27, Tainong 34, and Tainong 57 on a 15% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel was prevented by dipping the gels in solutions containing 10–40 mM hydrogen peroxide, 10 mM Tris buffer (pH 7.9) for 30 min before staining.
Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser‐induced flurescence detection
Authors: Klepárník Karel , Malá Zdeňka , Haváč Zdeněk , Blažková Michaela , Hollá Lydie , Boček Petr 
Published: May 2005

AbstractThe optimum separation conditions of polymerase chain reaction (PCR) products have been found for high‐speed capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection. DNA fragments obtained after PCR amplification of the region covering the (CA)18 microsatellite repeat in nitron 5 of the gene for FcERIβ, a high affinity glycoprotein receptor for IgE, located on chromosome 11 (11q13), were analyzed with the aim of investigating the repeat polymorphism. The results of polyacrylamide slab gel electrophoresis (PAGE), agarose gel electrophoresis, CE with absorbance detector and CE with LIF are compared. The CE with LIF proved to shorten analysis time by a factor of 100 when compared to slab gel electrophoresis. CE‐LIF utilizes a short capillary with an effective length of 6.3 cm and electric field strength from 100 to 550 V/cm. The respective PCR products of sizes from 116 to 210 base pairs (bp) were analyzed in 3 min.