bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparative proteomics of human endothelial cell caveolae and rafts using two‐dimensional gel electrophoresis and mass spectrometry
Authors: Sprenger Richard R. , Speijer Dave , Back Jaap Willem , de Koster Chris G. , Pannekoek Hans , Horrevoets Anton J. G. 
Published: January 2004

AbstractThe human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin‐enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two‐dimensional (2‐D) maps and subsequent identification of over 100 protein spots by matrix‐assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post‐translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold‐detergent method yielded a large number of known caveolae residents, including caveolin‐1. Thus, a large part of this subproteome was established, including known (trans‐)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)‐anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP‐2, C8ORF2, and XRP‐2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.
Profiling and comprehensive expression analysis of ABC transporter solute‐binding proteins of Bacillus subtilis membrane based on a proteomic approach
Authors: Bunai Keigo , Ariga Masanori , Inoue Taro , Nozaki Manabu , Ogane Shinya , Kakeshita Hiroshi , Nemoto Tadashi , Nakanishi Hiroshi , Yamane Kunio 
Published: January 2004

AbstractWe analyzed ABC transporter solute‐binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) or two one‐dimensional (1‐D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2‐D gel separation profiles of submembrane fractions solubilized by 1% n‐dodecyl‐β‐D‐maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn++, glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).
Quantitative and qualitative profiling of mitochondrial DNA length heteroplasmy
Authors: Lee Hwan Young , Chung Ukhee , Yoo Ji‐Eun , Park Myung Jin , Shin Kyoung‐Jin 
Published: January 2004

AbstractQuantitative and qualitative analysis of mitochondrial DNA length heteroplasmy for the first hypervariable segment (HV1) and second hypervariable segment (HV2) regions were performed using size‐based separation of fluorescently‐labeled polymerase chain reaction (PCR) products by capillary electrophoresis. In this report, the relative proportions of length heteroplasmies in individuals were determined, and each length variant in the heteroplasmic mtDNA mixture was identified. The study demonstrated that 36% and 69% of Koreans show length heteroplasmy in the HV1 and HV2 regions, respectively. Electropherograms revealed that length heteroplasmy in the HV1 region resulted in over 5 length variants in an individual. The peak patterns of length heteroplasmy in the HV1 region were classified into five major types. In the HV2 region, length heteroplasmy resulted in 3–6 length variants in an individual, and showed seven variant peak patterns. The increased knowledge concerning mtDNA length heteroplasmy is believed to not only offer a useful means of determining genetic identity due to increased mitochondrial DNA haplotype diversity by allowing mtDNAs to be classified into several peak patterns, but also represent a promising tool for the diagnosis of several common diseases which are etiologically or prognostically associated with mtDNA polymorphisms.
Resolution of 8‐aminonaphthalene‐1,3,6‐trisulfonic acid‐labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration
Authors: Cabanes‐Macheteau Marion , Chrambach Andreas , Taverna Myriam , Buzás Zsuzsanna , Berna Patrick 
Published: January 2004

AbstractA discontinuous Tris‐Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8‐aminonaphthalene‐1,3,6‐trisulfonic acid (ANTS)‐labeled malto‐oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris‐OAc behind a moving boundary formed between the leading chloride ion of Tris‐HCl gel buffer and the trailing OAc ion provided by a catholyte of NH4OAc. In contrast with the results obtained with Tris‐CI/glycinate buffer commonly used in electrophoresis, or with Tris‐CI/borate, the best resolution of the glucose oligomers containing 1–4 glucose units in Tris‐OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH4OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS‐glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, Rf, vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, KR, of the plots. The molecular weight of the oligomers is directly but nonlinearly related to KR. The novel electrophoretic system illustrated here for separation of short ANTS‐saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.
Alternative sample preparation prior to two‐dimensional electrophoresis protein analysis on solid lipid nanoparticles
Authors: Göppert Torsten M. , Müller Rainer H. 
Published: January 2004

AbstractThe proteins adsorbing onto the surface of intravenously injected drug carriers are regarded as a key factor determining the organ distribution. Depending on the particle surface properties, certain proteins will be preferentially adsorbed, leading to the adherence of the particle to cells with the appropriate receptor. Therefore, the knowledge of the protein adsorption pattern and the correlation to in vivo behavior opens the perspective for the development of intravenous colloidal carriers for drug targeting. After incubation in plasma, the adsorbed proteins were analyzed using two‐dimensional polyacrylamide gel electrophoresesis (2‐D PAGE, 2‐DE). The purpose of the present study was to develop an alternative separation method to separate solid lipid nanoparticles (SLN) carriers from plasma by gel filtration prior to 2‐D PAGE. Via the specific absorption coefficients and a two‐equation system, elution fractions were identified being practically plasma‐free. This allows protein analysis on SLN which are typically in density too close to the density value of water to be separated by the standard centrifugation method. The SLN used for establishing the gel filtration were prepared in a way that they had a sufficiently low density to be additionally separated by centrifugation. The adsorption patterns obtained after separation with both methods were qualitatively and quantitatively identical, showing the suitability of the gel filtration.
Feasibility of nonvolatile buffers in capillary electrophoresis‐electrospray ionization‐mass spectrometry of proteins
Authors: Eriksson Jonas H. C. , Mol Roelof , Somsen Govert W. , Hinrichs Wouter L. J. , Frijlink Henderik W. , de Jong Gerhardus J. 
Published: January 2004

AbstractThe combination of capillary electrophoresis (CE) and electrospray ionization‐mass spectrometry (ESI‐MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI‐MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5–10 μM (ca. 100 fmol injected). The usefulness of the CE‐MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE‐MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.
Zeta potential determination by streaming current modelization and measurement in electrophoretic microfluidic systems
Authors: Renaud Louis , Kleimann Pascal , Morin Pierre 
Published: January 2004

AbstractElectrophoresis in capillary and microfluidic systems, used in analytical chemistry to separate charged species, are quite sensitive to surface phenomena in terms of separation performances. In order to improve theses performances, new surface functionalization techniques are required. There is a need for methods to provide fast and accurate quantification about surface charges at liquid/solid interfaces. We present a fast, simple, and low‐cost technique for the measurement of the zeta‐potential, via the modelization and the measurement of streaming currents. Due to the small channel cross section in microfluidic devices, the streaming current modelization is easier than the streaming potential measurement. The modelization combines microfluidic simulations based on the Navier‐Stokes equation and charge repartition simulations based on the Poisson‐Boltzmann equation. This method has been validated with square and circular cross section shape fused‐silica capillaries and can be easily transposed to any lab‐on‐chip microsystems.
Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2
Authors: Suck Roland , Petersen Arnd , Weber Bernhard , Becker Wolf‐Meinhard , Fiebig Helmut , Cromwell Oliver 
Published: January 2004

AbstractSodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high‐resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N‐terminal sequences and IgE‐binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.
Study of the development of thermoresistance in human pancreatic carcinoma cell lines using proteome analysis
Authors: Poland Julia , Urbani Andrea , Lage Hermann , Schnölzer Martina , Sinha Pranav 
Published: January 2004

AbstractIn order to find candidate proteins that are potentially associated with the thermoresistant phenotype in combination with drug resistance, we analyzed the differential protein expression in vitro in the human pancreatic cancer cell line EPP85‐181‐P and classical and atypical multidrug‐resistant variants and their thermoresistant counterparts using proteomics. This study identifies sets of proteins that may lead to the development of thermoresistance. These results provide a fundamental basis to elucidate the molecular mechanism of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.
A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate‐polyacrylamide gels
Authors: Zhou Shaobo , Bailey Matthew J. , Dunn Michael J. , Preedy Victor R. , Emery Peter W. 
Published: January 2004

AbstractWe report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate‐polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70°C overnight followed by liquid scintillation counting. H2O2 had no effect on the count rates of [14C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70°C resulted in incomplete extraction of radioactivity from gels containing [14C]BSA, but there was also a significant reduction in count rates in samples incubated at 80°C. At 70°C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89–94%, and the coefficient of variation for five replicate samples was 5–10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein.
Direct ampholyte‐free liquid‐phase isoelectric peptide focusing: Application to the human serum proteome
Authors: Xiao Zhen , Conrads Thomas P. , Lucas David A. , Janini George M. , Schaefer Carl F. , Buetow Kenneth H. , Issaq Haleem J. , Veenstra Timothy D. 
Published: January 2004

AbstractIn this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid‐phase isoelectric focusing (IEF) in a novel ampholyte‐free format. Twenty IEF fractions were collected and analyzed by reversed‐phase microcapillary liquid chromatography (μLC)‐MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte‐free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high‐throughput characterization of proteins from complex proteomic mixtures.
Pyrosequencing™ technology at elevated temperature
Authors: Eriksson Jonas , Gharizadeh Baback , Nordström Tommy , Nyrén Pål 
Published: January 2004

AbstractTo date, the Pyrosequencing™ technology has been performed at 28°C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37°C. By increasing the temperature to 37°C, false signals due to primer‐dimers and loop‐structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza‐dGTP in the polymerase chain reaction (PCR), (ii) 1.6 M glycine betaine, and (iii) an increase of the temperature to 37°C enabled us to sequence a DNA template with the initial sequence 3'‐ATGGCCCGGGGGGGAGCTCCA … 5'. Furthermore, we describe a method to analyze if a primer forms a primer‐dimer with extendable 3'‐ends.
Vapor and liquid phase detection of cyanide on a microchip
Authors: Lu Qin , Collins Greg E. , Evans Thomas , Hammond Mark , Wang Joseph , Mulchandani Ashok 
Published: January 2004

AbstractA capillary electrophoresis microchip is used to selectively and sensitively monitor cyanide levels in both vapor (HCN(g)) and aqueous (NaCN in drinking water) phases. Laser‐induced fluorescence detection is applied using a violet diode laser to monitor the fluorescent isoindole derivative formed by the reaction of cyanide with 2,3‐naphthalenedicarboxaldehyde (NDA) and taurine. Air sampling of hydrogen cyanide is achieved using a miniature impinger (2 mL), giving collection efficiencies as high as 79% for a sampling rate of 1.0 L/min and a 10 s sampling time (relative standard deviation RSD: 2.7% for n = 5). Following the addition of NDA and taurine to either the vapor phase impinger sample or an aqueous drinking water sample, the NDA/cyanide derivative can be detected in just over 40 s on the microchip, giving a detection limit of 0.56 μg/L and a linear dynamic range from 0.56 μg/L–2.4 mg/L. The detection limit for hydrogen cyanide in air was determined to be 2.3 ppb (mole%). On‐chip derivatization of cyanide by NDA was successful, although a 50% decrease in signal intensity was observed due to insufficient time for completion of the reaction on the microchip. A number of different interferents were examined, and only iron(II) and chlorine showed any interference due to their capability for masking the presence of cyanide by reacting with free cyanide in solution.
Modelling and simulation of fires in vehicle tunnels
Authors: Gasser I. , Struckmeier J. , Teleaga I. 
Published: January 2004

AbstractApplying a low‐Mach asymptotic for the compressible Navier–Stokes equations, we derive a new fluid dynamics model,which should be capable to model large temperature differences in combination with the low‐Mach number limit. The model is used to simulate fires in vehicle tunnels, where the standard Boussinesq‐approximation for the incompressible Navier–Stokes seems to be inappropriate due to the high temperatures developing in the tunnel. The model is implemented using a modified finite‐difference approach for the incompressible Navier–Stokes equations and tested in some realistic fire events. Copyright © 2004 John Wiley & Sons, Ltd.
Newton linearization of the incompressible Navier–Stokes equations
Authors: Sheu Tony W. H. , Lin R. K. 
Published: January 2004

AbstractThe present study aims to accelerate the convergence to incompressible Navier–Stokes solution. For the sake of computational efficiency, Newton linearization of equations is invoked on non‐staggered grids to shorten the sequence to the final solution of the non‐linear differential system of equations. For the sake of accuracy, the resulting convection–diffusion–reaction finite‐difference equation is solved line‐by‐line using the proposed nodally exact one‐dimensional scheme. The matrix size is reduced and, at the same time, the CPU time is considerably saved due to the decrease of stencil points. The effectiveness of the implemented Newton linearization is demonstrated through computational exercises. Copyright © 2004 John Wiley & Sons, Ltd.
Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose
Authors: Bianco John I. , Perry Chris , Harkin Damien G. , Mackay‐Sim Alan , Féron François 
Published: January 2004

AbstractSeveral studies have demonstrated the potential of olfactory ensheathing cells for the repair of central and peripheral nerve injury. However, the majority of these studies have been performed with olfactory ensheathing cells derived from the olfactory bulbs, situated inside the skull. A more clinically relevant source of olfactory ensheathing cells is the olfactory mucosa, located in the nose. To be successful, an autologous transplant of nasal ensheathing glia would require a large number of purified cells. To address this issue, we have focused our research on three neurotrophic factors, namely nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3). We show here that their respective receptors, TrkA, TrkB, TrkC, as well as p75NTR (the low affinity NGF receptor), are expressed in vitro by the nasal ensheathing cells; the three neurotrophins promote purification and proliferation of these glial cells, with an optimal concentration of 50 ng/ml; and human ensheathing cells can be easily biopsied and highly purified using a serum‐free medium supplemented with NT3. This technique opens the door for clinical trials in which nasal ensheathing cells will be autotransplanted in humans suffering from nerve injury. © 2003 Wiley‐Liss, Inc.