bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of maize lines via capillary electrophoresis of zeins in isoelectric, acidic buffers
Authors: Righetti Pier Giorgio , Olivieri Erna , Viotti Angelo 
Published: May 2005

AbstractModified capillary zone electrophoresis of zeins in strongly acidic, isoelectric buffers is reported for screening of maize (Zea mays L.) lines. The optimized background electrolyte contained 40 mM aspartic acid (pH = pI = 2.77), 8 M urea and 0.5% short‐chain hydroxyethylcellulose (Mn 27000 Da) (apparent pH in 8 M urea: 3.9). Due to the low conductivity of such a buffer (0.7 mmhos), separations can be carried out at 800 V/cm, even in relatively large bore capillaries (50 μ ID), ensuring good sensitivity. The zein patterns thus obtained are species‐specific and allow easy identification of all maize lines tested. No adsorption of proteins to the silica wall is observed and high reproducibility in peak areas and transit times is obtained. The relatively slow migration time (ca. 30 min), even under such a high voltage gradient, is attributed to the paucity of basic amino acid residues typical of zeins, leading to an unfavorable charge/ mass ratio due to the modest net positive charge at the operative pH.
In vitro model system for the identification and characterization of proteins involved in inflammatory processes
Authors: Dax Claudia I. , Lottspeich Friedrich , Müllner Stefan 
Published: May 2005

AbstractAn in vitro model featuring important inflammatory cellular states was established, based on the murine monocyte/macrophage cell line RAW 264.7. Macrophages are key players in chronic inflammation, and major parts of the biochemical reactions taking place in vivo, e.g., the production of proinflammatory cytokines, can be triggered in vitro by stimulation of the cells with bacterial lipopolysaccharide (LPS). A mastergel, representing a synthetic image of the expressed basic set of cellular proteins, was designed by a computer‐assisted overlay of a statistically significant number of two‐dimensional electrophoresis (2‐DE) gels of unstimulated RAW 264.7 cells. This image served as a reference for qualitative and quantitative changes in the protein pattern induced by stimulation of the macrophages with LPS. The optimal conditions for LPS stimulation were evaluated by monitoring the expression and secretion of the proinflammatory cytokine tumor necrosis factor‐α (TNF‐α). The comparison of the mastergel with the 2‐DE gels of LPS‐stimulated cells revealed several changes in the protein pattern. In order to prove the relevance of the presented model system, we focused on two low molecular weight proteins, which showed significant changes in the apparent concentration in a 2‐DE pattern. These proteins were further characterized by microsequencing of internal peptides. A comparison of the obtained sequences with protein databases identified them as cofilin and keratinocyte lipid‐binding protein.
Glycobiology and proteomics: Is mass spectrometry the holy grail?
Authors: Packer Nicolle H. , Harrison Mathew J. 
Published: May 2005

AbstractOne characteristic of glycoproteins is that they are separated by two‐dimensional electrophoresis (2‐D PAGE) into typical ‘trains’ of protein spots which separate on the basis of different isoelectric point (pI) and/or molecular mass. The pattern of these trains often varies in development and disease. While the isoforms differ both in the number of sites of glycosylation and the types of carbohydrate attached to the protein, classical methods of glycan analysis are insensitive at the levels typically separated by 2‐D PAGE. Developments in mass spectrometry technologies have enabled the characterization of most of the oligosaccharide attributes to be determined on picomole amounts of protein. These techniques are beginning to allow the glycoform heterogeneity on 2‐D separated glycoproteins to be analyzed.
New zwitterionic detergents improve the analysis of membrane proteins by two‐dimensional electrophoresis
Authors: Chevallet Mireille , Santoni Véronique , Poinas Alexandra , Rouquié David , Fuchs Alexandra , Kieffer Sylvie , Kieffer Sylvie , Lunardi Joël , Garin Jérome , Rabilloud Thierry 
Published: May 2005

AbstractSevere quantitative loss of protein is often observed in high‐resolution two‐dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14–16 carbons proved most efficient, solubilizing previously undetected membrane proteins.
Suppression effects in enzymatic peptide ladder sequencing using ultraviolet ‐ matrix assisted laser desorption/ionization ‐ mass spectrometry
Authors: Kratzer Robert , Eckerskorn Christoph , Karas Michael , Lottspeich Friedrich 
Published: May 2005

AbstractThe techniques of enzymatic and chemical peptide ladder sequencing, coupled with ultraviolet ‐ matrix assisted laser desorption/ionization ‐ mass spectrometry (UV‐MALDI‐MS) have been improving continuously in the last five years and have now become important tools for primary structure identification. In this work, signal suppression effects, appearing in UV‐MALDI‐MS (excitation 337 nm) of ladder peptides, were investigated using the 17‐amino acid peptide dynorphin A. We show, with examples of simple “two‐peptide” systems and more complex “multi‐peptide” systems, that suppression effects do in fact exist. The magnitude of the observed suppression is strongly dependent upon both the nature and the amount of the suppressing peptide. Suppression behavior of individual ladder peptides was investigated on equimolar mixtures of ten ladder peptides. Significant signal suppression was recorded for all ladder peptides, with some of them being approximately 170 times lower in signal intensity than the pure, i.e., unsuppressed peptide at the same concentration. For the investigated system — dynorphin A, 4‐hydroxy‐α‐cyanocinnamic acid (4‐HCCA) matrix, UV excitation — a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
Towards an automated approach for protein identification in proteome projects
Authors: Traini Mathew , Gooley Andrew A. , Ou Keli , Wilkins Marc R. , Tonella Luisa , Sanchez Jean‐Charles , Hochstrasser Denis F. , Williams Keith L. 
Published: May 2005

AbstractThe development of automated, high throughput technologies for the rapid identification of proteins is essential for large‐scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation — time of flight (MALDI‐TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two‐dimensional gel electrophoresis (2‐DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI‐TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.
Strategies towards a better understanding of antibiotic action: Folate pathway inhibition in Haemophilus influenzae as an example
Authors: Evers Stefan , Padova Karin Di , Meyer Michelle , Fountoulakis Michael , Keck Wolfgang , Gray Christopher P. 
Published: May 2005

AbstractTwo‐dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.
Effects of renovascular hypertension on myocardial protein patterns: Analysis by computer‐assisted two‐dimensional gel electrophoresis
Authors: Pleißner Klaus‐Peter , Regitz‐Zagrosek Vera , Krüdewagen Bernd , Trenkner Jörg , Hocher Berthold , Fleck Eckart 
Published: May 2005

AbstractHypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer‐assisted two‐dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9‐week‐old rat siblings. Sham‐operated animals served as controls. Systolic blood pressure (197 ± 19 mm Hg) and heart/body weight ratios (0.36 ± 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 ± 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann‐Whitney test) as well as a multi‐variate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern.
Size separation of sodium dodecyl sulfate complexes of human plasma proteins by capillary electrophoresis employing linear polyacrylamide as a sieving polymer
Authors: Manabe Takashi , Oota Hideto , Mukai Jun 
Published: May 2005

AbstractElectrophoretic conditions to separate sodium dodecyl sulfate (SDS) complexes of human plasma proteins according to their size differences, by capillary electrophoresis employing linear polyacrylamide as a sieving matrix (LPA‐CE), have been examined. Using the optimized separation conditions, SDS complexes of human plasma proteins not treated with reducing agents were separated into about 40 peaks and shoulders within 60 min. The molecular mass values of major peaks in a separation pattern were estimated from a plot of molecular mass and migration time for standard proteins and some of the major plasma proteins have been identified on the pattern. The electrophoretic conditions were successfully applied for the analysis of proteins in immunoglo‐bulin G (IgG) myeloma sera.
Application of capillary electrophoresis, liquid chromatography, electrospray‐mass spectrometry and matrix‐assisted laser desorption/ionization ‐ time of flight ‐ mass spectrometry to the characterization of recombinant human erythropoietin
Authors: Zhou Guo‐Hua , Luo Guo‐An , Huang Le‐Qun , Zhou Yong , Zhang Xiao‐Dano , Zhou Ke‐Yu 
Published: May 2005

AbstractHigh performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC), matrix‐assisted laser desorption/ionization ‐ time of flight ‐ mass spectrometry (MALDI‐TOF‐MS), on‐line CE‐electrospray ionization‐mass spectrometry (CE‐ESI‐MS) and on‐line LC‐ESI‐MS have been employed to characterize a heterogeneous glycoprotein, recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The analysis was demonstrated through two specific levels of detail: the intact protein and tryptic digests of the protein. Six glycoforms of rHuEPO were separated by HPCE; seventeen tryptic fragments in a total of 21 nonglycosylated and glycosylated peptides were characterized; the O‐linked glycopeptides were analyzed directly by CE‐ESI‐MS and LC‐ESI‐MS. In particular, four glycans of O‐acetylation of sialic acid were identified in the O‐linked glycosylated fragments. The molecular weight of rHuEPO was accurately determined by MALDI‐TOF‐MS.
Multiplex polymerase chain reaction of Alu polymorphic insertions
Authors: Thomas Emmanuel , Herrera Rene J. 
Published: May 2005

AbstractAlu sequences are present in humans in excess of 500 000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. Polymorphic Alu insertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)‐based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to the Alu polymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five different Alu polymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimize Alu multiplex PCR.
Triple helix‐forming oligonucleotides which make imperfect Watson‐Crick duplexes that compete with the creation of the triplex
Authors: Porumb Horea , Gousset Hervé , Taillandier Eliane 
Published: May 2005

AbstractBecause of the repetitive nature of the sequence, when titrating a G,A‐rich, triple helix‐forming oligonucleotide (TFO) with increasing concentrations of target duplex in order to obtain the dissociation constant of the complex, a duplex is sometimes first generated at intermediate concentrations of the target. This duplex is based on an imperfect Watson‐Crick pairing of the TFO to the pyrimidine‐rich strand of the target. An explanation is proposed for this duplex being obtained only in a certain domain of the titration range, before the triple helix becomes predominant.
Development and comparison of assays for measuring immunospecific antibody response in serum of chickens and rabbits immunized with human immunoglobulin G
Authors: Bollen Lise S. , Hau Jann 
Published: May 2005

AbstractThe antibody response development during polyclonal antibody production is a relevant parameter to monitor during the immunization period to be able to optimize the immunization protocol and to determine the optimal antibody harvest time. Although rabbits and other mammals are most often used for polyclonal antibody production, the chicken is a relevant alternative. There are both scientific reasons, economic reasons, and animal welfare reasons to consider when choosing the chicken instead of a mammal for this purpose, because antibodies in generous quantities can be harvested from the egg yolk. This study compared different assays for measuring antibody response in rabbit and chicken serum. An inhibition liquid phase absorption assay (ILPAA), a rocket immunoelectrophoresis (RIE) assay, and a line immunoelectrophoresis (LIE) assay were compared to ELISAs. The ELISA proved to be the most useful assay for routine use, as it was less time‐consuming and because the assay could easily be adapted to both serum antibody types. However, electrophoretic assays were the most useful as combined analytical and quantitative tools and must be considered essential when analyzing specificities of polyclonal antibody preparations.
Boundary element modeling of electrokinetically driven fluid flow in two‐dimensional microchannels
Authors: Hoyt Jeffrey J. , Wolfer Wilhelm G. 
Published: May 2005

AbstractIn recent years there has been considerable intrerest in fabricating electrophoretic separation systems on microchips. In this study the boundary element method is used to numerically model both the electrical charge density and the electrokinetically driven fluid flow velocity field in two‐dimensional micro‐channels containing an arbitrary configuration of circular flow obstacles. An estimate of both the average velocity and the resolution has been determined for various obstacle configurations, obstacle sizes, area fractions, surface line lengths per unit area and for different values of the thickness of the electrical double layer. Based on the results, an optimal microchannel design is suggested. In addition, the recently observed phenomenon of recirculated flow in an open region of an otherwise packed electrochromatography column has been confirmed with the numerical model.
Use of ethanolamine for sample stacking in capillary electrophoresis
Authors: Louwagie Mathilde , Rabilloud Thierry , Garin Jérǒme 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) in the presence of ethanolamine was used in a micropreparative mode. Sample volumes up to 1 μL could be loaded onto a 100 μm diameter capillary without loss in resolution. Coupled to narrow‐bore reversed‐phase high‐performance liquid chromatography, ethanolamine‐CZE allowed the collection of sufficient amounts of pure peptidic material to perform amino acid sequence analysis.
High speed separation of DNA fragments by capillary electrophoresis in poly(ethylene oxide)‐poly(propylene oxide)‐poly(ethylene oxide) triblock polymer
Authors: Liang Dehai , Chu Benjamin 
Published: May 2005

AbstractThe high speed separation of DNA fragments by using a triblock copolymer, 25% w/v F127 (PEO99PPO69PEO99 with PEO and PPO denoting polyethylene oxide and polypropylene oxide, respectively) which is easy to handle and does not need coating of the quartz capillary, has been investigated. Two ways to decrease the run time are presented: one is to shorten the effective capillary length and the other to increase the electric field strength. In a short capillary, the sieving ability of the separation medium versus the initial band width, and the band width spreading as a function of distance traveled dominate the resolution; at high electric field strength, Joule heating could deteriorate the separation. By taking both effects into account, the ΦX174/HaeIII digest could be separated within 100s by using an 8 mm effective length, 50 μm diameter capillary operating at 300 V/cm.
Preparative capillary zone electrophoresis in combination with off‐line graphite furnace atomic absorption for the analysis of DNA complexes formed by a new aminocoumarine platinum (II) compound
Authors: Deforce Dieter L. D. , De Leenheer Andreas , Van Den Eeckhout Elfriede G. , Kokotos George , Esmans Eddy L. 
Published: May 2005

AbstractCalf thymus DNA was incubated in vitro with a new aminocoumarin platinum(II) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5′‐mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5′‐mononucleotides.
Particle electrophoresis of micrometric‐sized superparamagnetic particles designed for magnetic purification of cells
Authors: Sestier Claude , Sabolovic Domagoj 
Published: May 2005

AbstractMeasuring the electrophoretic mobility of superparamagnetic particles (0.5–4.5 μm mean size) was undertaken to probe the coupling of lectins and antibodies to their surface. Coupling was either noncovalent (antigen‐antibody and biotinstreptavidin linkage) or covalent (tosyl‐activated beads). The direct observation of the electrophoresis of single particles illuminated in dark field and processed by image analysis allowed the determination of their apparent electrophoretic mobility. Mobilities ranged from −0.5 μm s−1/CmV−1 to + 1 μm s−1/CmV−1 when measured at 20°C in 0.15 M NaCl and 30 mg/mL sorbitol, pH 7.4. The relative standard deviation was less than 0.1%. Surface immobilization of charged proteins onto the superparamagnetic beads shifted their electrophoretic mobility up to 200%; this was also quantitatively correlated with some specific properties (enzymatic activity, antigen‐binding activity, lectin‐binding activity). Although particle electrophoresis has mainly been reported for the study of surface adsorption phenomenon, it may be a versatile tool for controlling covalent modifications of particles designed for therapeutical targeting or chromatographic use and may also apply to a quantitative analysis of ligand‐binding interactions.
Determination of glucose by capillary electrophoresis/laser‐induced fluorescence in transdermally collected samples
Authors: Frerichs Valerie , Colón Luis A. 
Published: May 2005

AbstractCapillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection has been used for the determination of glucose in samples collected by noninvasive means. The method uses an enzymatic reaction scheme that provides for the determination of small quantities of glucose with detection limits of 80 nM. This approach is used to evaluate passive transdermal diffusion as a noninvasive means to sample glucose in vivo. A simple sampling cell design is presented. Sample collection was performed on volunteer human subjects. Our experiments show that fluctuations in blood glucose concentration are reflected in the samples obtained by passive transdermal diffusion after glucose intake. The results indicate that glucose from the subcutaneous fluid can be accessed by passive diffusion.