bluuebunny/crossref_metadata_2025_split huggingface dataset:
Natural resistance to intracellular parasites: A study by two‐dimensional gel electrophoresis coupled with multivariate analysis
Authors: Kovářová Hana , Radzioch Danuta , Hajdúch Marián , Šírová Milada , Bláha Václav , Macela Aleš , Stulík Jiří , Hernychová Lenka 
Published: May 2005

AbstractNatural resistance to Mycobacterium bovis bacillus Calmette‐Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two‐dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R‐Rb macrophages transfected with Nrampl‐ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele‐carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R‐Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn‐superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl‐2 according to the immunoblot analysis. When the splenic macrophages carring the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.
Amplification of microsatellites adapted from human systems in faecal DNA of wild Hanuman langurs (Presbytis entellus)
Authors: Launhardt Kristin , Epplen Cornelia , Epplen Jörg T. , Winkler Paul 
Published: May 2005

AbstractMicrosatellite systems originally established for human DNA were utilized for paternity testing from faecal DNA in a natural population of Hanuman langurs (Presbytis entellus). Thirty‐two primer pairs were applied to amplify DNA obtained from langur faeces. Twenty‐two of these primer pairs yielded specific amplification products and 11 loci were polymorphic. Allele distributions and heterozygosity rates were determined for five systems. Genetic information from these five systems was sufficient for paternity exclusion in 46 out of 52 cases. Results were consistent enough to allow genotyping from faeces, although sometimes only one allele was amplified in a heterozygous individual. In conclusion, relationship analyses from faeces are possible in spite of the evolutionary distance between humans and langurs.
The effect of complexation additives on analyte migration behavior in capillary electrochromatography
Authors: Bowser Michael T. , Chen David D. Y. 
Published: May 2005

AbstractIn capillary electrochromatography (CEC), analytes often have different mobilities in the mobile phase, and often are involved in multiple equilibria. In this paper, the migration behavior of an analyte in CEC is described by a general equation in which individual capacity factors are used to describe the tendency of the analyte to exist as the various analyte species present in a separation system, and the effects of both field and equilibrium are accounted for. The resolution of two analytes is shown to be related linearly to the ratio of their migration rates. The effect of the electroosmotic flow (EOF) in CEC is more complicated than in CE because it is experienced only by a fraction of the analyte, whereas in CE, it is experienced by all analyte species. A procedure for calculating the electrophoretic mobility of the analyte based on the fraction of the analyte in the buffer is demonstrated. The effect of the EOF on resolution is also discussed.
Two‐dimensional electrophoresis of proteins in an immobilized pH 4–12 gradient
Authors: Görg Angelika , Boguth Günther , Obermaier Christian , Weiss Walter 
Published: May 2005

AbstractFor checking theoretical two‐dimensional (2‐D) maps derived from sequenced genomes, indicating that nonnegligible amounts of proteins up to pH 12 are to be expected, a wide‐range immobilized pH 4–12 gradient was generated. Depending on the extraction method of sample preparation, proteins with pIs up to pH 12 are detected in a single gel. Highly reproducible protein patterns focused to the steady state with round‐shaped spots up to pH 12 are obtained with the standard protocol originally described in 1988 (Görg et al., Electrophoresis 1988, 9, 531–546).
Recent developments in DNA electrophoretic separations
Authors: Slater Gary W. , Kist Tarso B. L. , Ren Hongji , Drouin Guy 
Published: May 2005

AbstractDNA electrophoresis is now a fairly mature technology. Nevertheless, as we approach the 21st century, new ideas are frequently suggested that could lead to a revolution for DNA sequencing and mapping. Here, we review some of the novel concepts that have been studied since ca. 1990. Our review focuses on new separation mechanisms, new sieving matrices and recent conceptual advances.
Human globin chain separation by capillary electrophoresis in acidic isoelectric buffers
Authors: Righetti Pier Giorgio , Saccomani Arianna , Stoyanov Alexander V. , Gelfi Cecilia 
Published: May 2005

AbstractA simple and reliable method, utilizing capillary electrophoresis in uncoated capillaries in acidic isoelectric buffers, is reported for screening for thalassemia and other defects on the synthesis of human globin chains. A solution of 50 mM iminodiacetic acid (pi2.23), containing 7 M urea and 0.5% hydroxyethyl‐cellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme‐free, denatured globin (α, β and γ) chains. Due to the low conductivity of such a buffer, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. It is additionally shown that inclusion of 2% surfactant (Tween 20) in the background electrolyte induces the splitting of the γ chains into two zones, called Aγ and Gγ, which represent the products of two genes coding for Ala or Gly as residue 136 of the chain.
Electrophoresis combined with novel mass spectrometry techniques: Powerful tools for the analysis of proteins and proteomes
Authors: Figeys Daniel , Gygi Steven P. , Zhang Yanni , Watts Julian , Gu Ming , Aebersold Ruedi 
Published: May 2005

AbstractAnalytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)‐MS/MS, capillary‐HPLC‐MS/MS, and solid phase extraction (SPE)‐capillary zone electrophoresis (CZE)‐MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome‐wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE‐CZE‐MS/MS, and IMAC followed by HPLC, and by SPE‐CZE‐MS/MS, are presented for the detailed investigation of post‐translational modifications of specific proteins.
Reference map of the low molecular mass proteins of Haemophilus influenzae
Authors: Fountoulakis Michael , Juranville Jean‐François , Röder Daniel , Evers Stefan , Berndt Peter , Langen Hanno 
Published: May 2005

AbstractAnalysis of the proteome of Haemophilus influenzae by two‐dimensional polyacrylamide gel electrophoresis on conventional Tris‐glycine gels does not usually result in efficient separation of the proteins in the 5–20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second‐dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5–25 kDa region were identified by matrix assisted laser desorption/ionization ‐ mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys‐C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.
Capillary electrochromatography of derivatized mono‐ and oligosaccharides
Authors: Yang Changming , Rassi Ziad El 
Published: May 2005

AbstractAn octadecyl‐silica (ODS) stationary phase with light surface coverage of octadecyl ligands was introduced for capillary electrochromatography (CEC) at moderate electroosmotic flow (EOF) velocity. The ODS stationary phase was intentionally produced with light surface coverage in order to ensure a moderate EOF velocity across the packed capillary column, thus allowing relatively rapid analysis time. Despite the fact that the stationary phase leaves 75% of the surface silanols unreacted, fused‐silica capillary columns packed with this ODS stationary phase exhibited reversed‐phase behavior toward neutral alkylbenzene homologous solutes using hydroorganic eluents. Closely related p‐nitrophenylglycosides including some p‐nitrophenyl‐monosaccharides and p‐nitrophenyl‐maltooligosaccharides were readily separated on the ODS capillary column within a relatively short analysis time. Also, α‐ and β‐anomers of some p‐nitrophenyl‐monosaccharides were readily separated in the presence of a small amount of borate buffer in the hydroorganic eluent.
Capillary electrochromatography with novel stationary phases. I. Preparation and characterization of octadecyl‐sulfonated silica
Authors: Zhang Minquan , Rassi Ziad El 
Published: May 2005

AbstractA novel silica‐based stationary phase was developed for use in capillary electrochromatography (CEC) at relatively high electroosmotic flow (EOF). The silica was first bonded with a relatively hydrophilic layer bearing strong sulfonic acid groups. To this charged polar sublayer, octadecyl functions were covalently attached to yield the nonpolar top layer. This novel stationary phase, referred to as octadecylsulfonated silica (ODSS), was packed in bare fusedsilica capillaries or in capillaries with the same coating as the sublayer on the silica‐based stationary phase. The resulting packed columns were evaluated in CEC using alkylbenzenes as the test model solutes. Good separations can be achieved in less than 8 min, much faster than when using a regular octadecyl silica capillary column. Due to the permanent negative charge provided by the sulfonated sublayer on both the capillary walls and the silica particles, the magnitude of the EOF remained more or less constant over a wide range of pH, and its magnitude can be conveniently varied by the applied voltage.
Pressure‐assisted and pressure‐programmed capillary electrophoresis/electrospray ionization time of flight ‐ mass spectrometry for the analysis of peptide mixtures
Authors: Cao Ping , Moini Mehdi 
Published: May 2005

AbstractPressure assisting and pressure programming the inlet of the capillary electrophoresis instrument were used for the analysis of peptide mixtures and protein digests using capillary electrophoresis/electrospray ionization ‐ mass spectrometry (CE/ESI‐MS). CE/ESI‐MS of peptide mixtures and tryptic digests of proteins was studied using three different types of capillary columns: (i) a freshly aminopropylsilane (APS)‐treated column, (ii) an untreated column, and (iii) a degraded APS‐treated column. To maintain a constant and adequate buffer flow toward the CE capillary outlet for stable CE and ESI operation, low pressure was applied to the inlet of the CE when an untreated or degraded APS capillary was used. By programming the inlet pressure, CE/ESI‐MS analysis time was reduced to 1/3 of its original time. The utility of this technique is demonstrated by CE/ESI‐MS analysis of a hemoglobin variant (hemoglobin‐S) and its tryptic digests. Identification of the mutant peptide in the tryptic digest of hemoglobin‐S was achieved by collision‐induced dissociation (CID) of the protein digests using CE/ESI time of flight ‐ mass spectrometry (TOF‐MS).
Capillary zone electrophoresis of proteins with fused‐silica capillaries having polymers and surfactants adsorbed onto surfactant moieties previously covalently bound to the capillary column surface
Authors: Yang Changming , Rassi Ziad El 
Published: May 2005

AbstractFused‐silica capillary columns having cationic surfactant moieties (CSM) covalently attached to the capillary inner walls were introduced for the separation of proteins by capillary zone electrophoresis (CZE). The CSM capillary coating proved to be useful in the separation of basic and acidic proteins when modified hydroxypropylcellulose (HPC), namely epoxybutane‐HPC (EBHPC), was adsorbed to the primary CSM coating, yielding a hybrid coating (i.e., coating consisting of a covalenty bound ligand and an adsorbed ligand). The EBHPC layer rendered the capillary surface highly hydrophilic, thus permitting the separation of basic proteins with relatively high plate counts. Also, the CSM coating was useful for the separation of acidic proteins when the capillary wall had, in addition, a negatively charged polymer adsorbed to the surface, e.g., hyaluronic acid. In general, neutral and charged polymeric compounds could be readily adsorbed by the CSM coating, thus altering the zeta potential of the capillary and diminishing solute adsorption to the capillary surface. In other words, the sign of the zeta potential of the capillary surface could be tailored to be of the same sign as the charge of the analytes. Under these conditions, little or no solute‐wall interaction could be observed due to electrostatic repulsion. Although the capillary surface was charged, the presence of adsorbed polymer suppressed or, in most cases, even eliminated the electroosmotic flow (EOF).
Comparison of protein mixtures in aqueous humor by membrane preconcentration ‐ capillary electrophoresis ‐ mass spectrometry
Authors: Rohde E. , Tomlinson Andy J. , Johnson D. H. , Naylor Stephen 
Published: May 2005

AbstractThe significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1‐ and 2‐dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on‐line capillary electrophoresis — mass spectrometry (CE‐MS) for this application. Specifically, we used membrane preconcentration‐CE‐MS (mPC‐CE‐MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1) μL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS‐PROT database using Tagldent from ExPaSy. Among those proteins tentatively identified are β‐2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (β‐2 microglobulin), 13400–14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and ∼68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the α‐and β‐chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC‐CE‐MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.
Maturation of phagosomes is accompanied by specific patterns of small GTPases
Authors: Meyer Markus , Mayer Thomas , Tiedtke Arno 
Published: May 2005

AbstractIn this study we purified phagosomes of the ciliated protozoan Tetrahymena thermophila to analyze aspects of the maturation pathway of phagocytotic vesicles. Phagosomes were labeled with magnetic microparticles and then purified in high amounts with the help of a permanent magnet. By combining a pulse‐chase labeling protocol with the magnetic separation procedure we were able to isolate phagosomes of defined ages, which represent distinct stages of their maturation pathway. GTP‐overlay assays showed that a set of small GTPases of the ras superfamily is associated with these phagosomes. Phagosomes isolated at different stages of maturation revealed a change in the pattern of the small GTPases. Some small GTPases identified by the GTPase overlay assays could be aligned to India ink stained protein spots in two‐dimensional gels of isolated phagosomes. The results presented here are a first step to identify the members of small GTPases associated with phagosomes during their maturation pathway. Microsequencing of pooled polypeptides by mass‐spectrometric techniques will identify the primary structure of these small GTPases.
A tabulated review of capillary electrophoresis of carbohydrates
Authors: Suzuki Shigeo , Honda Susumu 
Published: May 2005

AbstractThis review summarizes publications on capillary electrophoresis (CE) of carbohydrates, covering almost all hitherto published papers on this topic. It is designed to be a convenient tool for the literature search by providing a comprehensive table. Since CE analysis of carbohydrates is generally complicated due to the structural diversity of carbohydrate species, an attempt is made in this table to supply detailed information on the analyzed form (underivatized or derivatized, type of derivative) and analytical conditions (capillary size, state of the inner wall, composition of the electrophoretic solution, applied voltage, detection method, etc.), for each combination of carbohydrate species to be analyzed. In addition, a brief overview is presented to help in the literature search.
Separation of disaccharides by affinity capillary electrophoresis in lectin‐containing electrophoretic solutions
Authors: Taga Atsushi , Yabusako Yukiko , Kitano Atsuko , Honda Susumu 
Published: May 2005

AbstractSeparation of the 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP) derivatives of simple disaccharides (maltose, cellobiose, gentiobiose, lactose, and melibiose) by affinity capillary electrophoresis was investigated using lectin‐containing neutral phosphate buffers, filled in a linear polyacrylamide‐coated capillary. When Lens culinaris agglutinin (LCA) was added, the derivatives of glucobioses were retarded with varying magnitudes depending on the amount of LCA and were well separated from each other and from galactosyl glucose under optimized conditions. Addition of Ricinus communis 60 kDa agglutinin (RCA60) to the phosphate buffer gave a different migration profile, in which the derivatives of galactosyl glucoses were more retarded than those of glucobioses. However, addition of either lectin did not accomplish complete separation of the derivatives of all these disaccharides even under optimum conditions. The addition of two kinds of lectins in appropriate proportions improved separation. Thus, the binary system composed of LCA and RCA60, as well as LCA and soybean agglutinin from Glycine max (SBA), gave better separation of these derivatives, giving peak tops for all derivatives.
Mapping and identification of Corynebacterium glutamicum proteins by two‐dimensional gel electrophoresis and microsequencing
Authors: Hermann Thomas , Wersch Gregor , Uhlemann Eva‐Maria , Schmid Roland , Burkovski Andreas 
Published: May 2005

AbstractAs a prerequisite for proteome analyses of Corynebacterium glutamicum separation of the cytoplasm and the membrane fraction was optimized and two‐dimensional (2‐D) gel electrophoresis was established. The resulting 2‐D protein maps revealed over 1000 silver‐stained protein spots separated by isoelectric point and molecular mass for cytoplasmic proteins and approximately 700 silver‐stained spots for proteins of the membrane fraction. Proposing a mean size of 1 kbp per gene the complete C. glutamicum genome of 3 Mbp encodes 3000 different proteins; more than half of these can be located using the maps which are presently available. In this study 10 proteins were identified by N‐terminal microsequencing, namely the 35 kDa antigen, antigen 84, ATP synthase subunits α, γ and δ, cysteine synthase, elongation factor G and Ts, enolase, and rotamase. For seven sequences, corresponding proteins could not be identified. Additionally, two proteins were specifically detected by immunoblotting, a corynebacterial porin and the cytoplasmic protein threonine dehydratase. The methods and 2‐D maps established in this study will be the basis for comparative studies of protein expression and a detailed proteome analysis of C. glutamicum.
On the use of generalized linear models for interpreting climate variability
Authors: Chandler Richard E. 
Published: May 2005

AbstractMany topical questions in climate research can be reduced to either of two related problems: understanding how various different components of the climate system affect each other, and quantifying changes in the system. This article aims to justify the addition of generalized linear models to the climatologist's toolkit, by demonstrating that they offer an intuitive and flexible approach to such problems. In particular, we provide some suggestions as to how ‘typical’ climatological data structures may be represented within the GLM framework. Recurring themes include methods for space–time data and the need to cope with large datasets. The ideas are illustrated using a dataset of monthly U.S. temperatures. Copyright © 2005 John Wiley & Sons, Ltd.