bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative analysis of hyaluronan in human synovial fluid using capillary electrophoresis
Authors: Grimshaw James , Trocha‐Grimshaw Jadwiga , Fisher William , Rice Aislinn , Smith Susan , Spedding Peter , Duffy John , Mollan Raymond 
Published: May 2005

AbstractThe glycosaminoglycan, hyaluronan, can be detected in human synovial fluid by capillary electrophoresis (CE). Variations in peak shape make this technique unsuitable for quantitative analysis of hyaluronan in raw synovial fluid. Quantitative analysis was achieved by hydrolysis of the polymeric hyaluronan to the tetrasaccharide by the action of testicular hyaluronidase and separation of the product using CE. A UV detector operating at 200 nm was used. The X‐ray contrast material, omnipaque, a propriety aqueous solution of iohexol was used as internal standard. A second peak in the electropherogram of synovial fluid was quantified. The variation in concentrations of these two components correlate with the arthritic disease state of a joint.
High‐speed separation of subnanomolar concentrations of noradrenaline and dopamine using capillary zone electrophoresis with laser‐induced fluorescence detection
Authors: Bert Lionel , Robert Frédéric , Denoroy Luc , Renaud Bernard 
Published: May 2005

AbstractCapillary zone electrophoresis with laser‐induced fluorescence detection has been shown to give rapid separations with high resolution and sensitivity. These advantages are documented for catecholamines analysis in the present work, which shows that separation of subnanomolar concentrations of dopamine and noradrenaline (detection limits of 8.6 × 10−11 M, corresponding to a detected amount of 143 zeptomoles of each catecholamine) can be performed in 82 s with an efficiency of several million theoretical plates.
Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: III. DNA separations by capillary electrophoresis in poly(N‐acryloylaminopropanol)
Authors: Gelfi Cecilia , Simò‐Alfonso Ernesto , Sebastiano Roberto , Citterio Attilio , Righetti Pier Giorgio 
Published: May 2005

AbstractSeparation of DNA fragments in a novel polymer network, consisting of N‐acryloylaminopropanol (AAP) is reported. The performance of this novel monomer, as a sieving liquid polymer in capillary zone electrophoresis, was evaluated. In 50 μm ID capillaries, an 8% solution of poly(AAP) can afford apex‐resolution of the 123/124 bp adjacent pair of DNA fragments in marker V, typically unresolved in any poly(acrylamide) formulation. It is proposed that the distal –OH group in the AAP molecule can form transient H‐bonds with the DNA double helix. Molecular modeling shows a meandering structure for poly(AAP), lacing the walls of half a cylinder, with kinks protruding at regular intervals, potentially able to fit into the major groove of DNA. Contrary to previously held beliefs, there seems to be a minimum length of the polymer for proper sieving of DNAs. For poly(acrylamides), a weight average molecular mass Mw 30 000 polymer offers no resolution, whereas a polymer of 250 000 to 400 000 Da exhibits optimum resolving power. For poly(AAP), the optimal length is in excess of 450 000 Da in Mw. Thus, it is shown that both the chemical composition of the monomer and the length of the polymer play a subtle, cooperative role in DNA separation.
The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis
Authors: Yarmola Elena , Sokoloff Helen , Chrambach Andreas 
Published: May 2005

AbstractDNA of approximately 2 kbp in length was previously found not to diffuse significantly in 1–1.5% agarose gels in the absence of an electric field, but to disperse during electrophoresis (Yarmola, E., Chrambach, A., Electrophoresis 1995, 16, 345–349). Accordingly, a process distinct from diffusion, and responsible for band spreading with migration time in gel electrophoresis, was defined as dispersion. Correspondingly, the diffusion coefficient, D(diff), was distinguished from a dispersion coefficient, D(disp). For DNA of approximately 1, 2 and 3 kbp, D(diff) and D(disp) were measured in agarose gel electrophoresis (1.0% SeaKem GTG). In that order of DNA length, D(disp) / D(diff) was found to increase from 5 to 15 to 45, showing that with increasing DNA length, time‐dependent band spreading, and thus resolution in gel electrophoresis, is governed predominantly by dispersion, not diffusion. It is assumed that the essential part of electrophoretic dispersion is due to entanglement of the DNA molecule in the gel. Indirect evidence for such an entanglement derives from the observation of peak asymmetry and its interpretation by the Giddings‐Weiss model.
Rapid loading of large sample volumes, analyte cleanup, and modified moving boundary transient isotachophoresis conditions for membrane preconcentration‐capillary electrophoresis in small diameter capillaries
Authors: Tomlinson Andy J. , Benson Linda M. , Jameson Stephen , Naylor Stephen 
Published: May 2005

AbstractUsing a removable membrane preconcentration (mPC) cartridge, large sample volumes can be loaded prior to final assembly of the mPC capillary electrophoresis (CE) capillary. For narrow‐bore (⩽ 25 μm ID) uncoated mPC‐CE capillaries, applied to peptide analysis, efficient moving boundary transient isotachphoresis (tITP) conditions at the onset of electrophoresis are described. The enhancement of mPC‐CE‐mass spectrometry (MS) technology afforded by rapid sample loading and modified moving boundary tITP conditions are demonstrated by analysis of major histocompatibility complex (MHC) class I peptides that were derived from a Kb precipitation of mouse EL‐4 cells. Furthermore, we demonstrate the structural characterization of these immunologically significant molecules by mPC‐CE‐tandem mass spectrometry (mPC‐CE‐MS/MS).
A kinetic study of the methanolysis of the sulfonylureas bensulfuron methyl and sulfometuron methyl using capillary electrophoresis
Authors: Matchett William H. , Winnik Witold , Betowski Leon O. , Brumley William C. 
Published: May 2005

AbstractThe instability of sulfonylureas in solution in methanol has led us to a kinetic study of methanolysis of two sulfonylureas using capillary electrophoresis. In a preliminary experiment, solutions of the seven compounds, bensulfuron methyl, sulfometuron methyl, nicosulfuron, chlorimuron ethyl, thifensulfuron methyl, metsulfuron methyl, and chlorsulfuron were prepared in methanol and in acetonitrile. After six weeks at room temperature the compounds dissolved in acetonitrile were quite stable as shown by electropherograms obtained with free zone capillary electrophoresis (CE). All seven of the compounds dissolved in methanol had undergone extensive degradation and in some cases the sulfonylurea could not be detected. Bensulfuron methyl and sulfometuron methyl were selected for further detailed study. Solutions of these two compounds in methanol were heated at 5, 40, 50, 57, and 65°C. No decomposition of either compound was observed in similar solutions of acetonitrile incubated at 57°C. The rates of decomposition in methanolic solutions were estimated from the decrease in instrumental response representing the peaks of the two sulfonylureas. The methanolysis of both compounds proceeded with pseudo first‐order kinetics as evidenced by the fact that semilogarithmic plots of the concentration of the substrate as a function of time were linear (correlation coefficients of 0.99). Rate constants and half‐lives for the reactions at 40, 50, 57, and 65°C were determined. These varied by factors of 18 to 22 depending upon the reaction temperature. The energies of activation for these reactions were estimated from the appropriate Arrhenius plots and found to be 26 kcal per mole for bensulfuron methyl and 24.5 kcal per mole for sulfometuron methyl. The kinetics of appearance of the products of methanolysis were directly related to and accurately predicted by the pseudo first‐order rate constants observed for disappearance of the sulfonylureas. Kinetic study of the reaction mixture by negative and positive‐ion mass spectrometry indicated that the products of methanolysis of sulfometuron methyl were 2‐carboxymethyl (N‐carboxymethyl)benzsulfonamide and 2‐amino‐4,6‐dimethyl pyrimidine and suggested a general mechanism for methanolysis of each of the seven compounds studied.
Prefractionation of protein samples prior to two‐dimensional electrophoresis
Authors: Corthals Garry L. , Molloy Mark P. , Herbert Ben R. , Williams Keith L. , Gooley Andrew A. 
Published: May 2005

AbstractThousands of proteins may be visualised on a two‐dimensional (2‐D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2‐D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub‐picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2‐D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the ‘reflux’ mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of specific proteins.
Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry
Authors: Dainese Paola , Staudenmann Werner , Quadroni Manfredo , Korostensky Chantal , Gonnet Gaston , Kertesz Michael , James Peter 
Published: May 2005

AbstractRecently the determination of the genome sequences of three procaryotes (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma genitalium) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were completed. Between 40–60% of the genes were found to code for proteins to which no function could be assigned. We describe an approach which combines proteome analysis (mapping of expressed proteins isolated by two‐dimensional polyacrylamide gel electrophoresis to the genome) with genetic manipulations to study the complex pattern of protein regulation occurring in Escherichia coli in response to sulfate starvation. We have previously described the upregulation of eight spots on two‐dimensional (2‐D) gels in response to sulfate starvation and the assignment of six of these to entries in the E. coli genome sequence (Quadroni et al., Eur. J. Biochem. 1996, 239, 773–781). Here we describe the identification of the remaining two proteins which are encoded in a sulfate‐controlled operon in the 21.5′ region of the E. coli genome. Upregulated protein spots were cut from multiple 2‐D gels collected and run on a modified funnel gel to concentrate the proteins and remove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database or a six‐frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post‐source decay in a matrix‐assisted laser desorption ionization (MALDI) time‐of‐flight mass spectrometer or collision‐induced dissociation (CID) coupled mass specrometry (MS/MS) with triple stage quadrupole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the SwissProt and EMBL databases.
Specific sample preparation in colorectal cancer
Authors: Reymond Marc A. , Sanchez Jean‐Charles , Schneider Claus , Rohwer Peter , Tortola Silvia , Hohenberger Werner , Kirchner Thomas , Hochstrasser Denis F. , Köckerling Ferdinand 
Published: May 2005

AbstractLarge tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti‐cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two‐dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 × 107 viable normal and tumoral cells before fixation, and up to 4 × 106 cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin‐positive cells in the samples was the following (mean, Cl 5–95%): mucosa 29.5% (8.9–66.7%), tumor 44.3% (6.6–94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.
Five cases of forensic short tandem repeat DNA typing
Authors: Schmitt Cornelia , Benecke Mark 
Published: May 2005

AbstractIn medicolegal samples DNA is often broken into fragments. In many cases, only the amplification of short tandem repeated DNA stretches (STRs), which are located in noncoding regions, allows DNA typing of such degraded materials. To demonstrate the high diversity of biological materials which forensic biologists have to deal with, and to outline the success rates and limits of the method, we describe five cases (minute amount of tissue on barrel, tissue in decay, tumor tissue, sperm after multiple rape, stored urine samples) in which forensic DNA typing was successfully performed by use of the short tandem repeats HUMDHFRP2, HUMD8S306, HUMCD4, HUMF13A1, HUMTH01, HUMVWA, and HUMFESPresented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.
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Resolution of acylated dipeptide stereoisomers by capillary electrophoresis using sulfobutylether derivatized β‐cyclodextrin
Authors: Skanchy David J. , Wilson Richard , Poh Tan , Xie Gaung‐H. , Demarest Charles W. , Stobaugh John F. 
Published: May 2005

AbstractThe separation of enantiomerically and diastereomerically related stereoisomers of acylated Asp‐Phe dipeptides was explored using capillary electrophoresis (CE). This series of dipeptides included the α‐L,L parent compound and the three other potential Asp containing stereoisomers (α‐D,D, α‐L,D, and α‐D,L), as well the four possible isoAsp containing stereoisomers (β‐L,L, β‐D,D, β‐L,D and β‐DL). The separation of these substances was explored using both neutral and charged cyclodextrins as the stereoisomer selector added to the running electrolyte. The major experimental parameters investigated included pH, the cyclodextrin type, and the cyclodextrin concentration. Due to differences in the pKa values of the carboxylic acid groups, adjustment of the separation buffer to between pH 3.0 and 4.0 provided for sufficient electrophoretic mobility differences to result in excellent separations of the diastereomerically related peptides in this pH region. The resolution of the enantiomerically related peptide stereoisomers was accomplished using low concentrations (1 mM) of the anionic cyclodextrin derivative, sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). This negatively charged cyclodextrin was found to be superior for the resolution of the enantiomerically related peptides as compared to native β‐cyclodextrin or the neutral derivatives, dimethyl β‐cyclodextrin and hydroxypropyl β‐cyclodextrin. An alternative approach using anionic or neutral surfactants in conjunction with the SBE‐β‐CDs was also explored and found to be successful but problematic.
Identification and classification of autoantibody repertoires (Western blots) with a pattern recognition algorithm by an artificial neural network
Authors: Grus Franz‐Hermann , Zimmermann Christoph W. 
Published: May 2005

AbstractThe screening of sera for autoantibodies with Western blots reveals complex repertoires. The composition of such repertoires depends on genetic control of autoantibody‐producing cells, the individual's history of exposure to its own and to foreign antigens, and also on the presence of autoimmune diseases. Our method shows how staining patterns of Western blots can be recoded as binary or grey‐value vectors. Vectors are transferred to artificial neural networks for learning. Artificial neural networks are able to recognize group‐specific antibody binding patterns. Staining patterns can be attributed to diagnostic groups. This may support diagnostic procedures.
The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products
Authors: Issaq Haleem J. , Chan King C. , Muschik Gary M. 
Published: May 2005

AbstractThis work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2–7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.
Proteome research: Complementarity and limitations with respect to the RNA and DNA worlds
Authors: Humphery‐Smith Ian , Cordwell Stuart J. , Blackstock Walter P. 
Published: May 2005

AbstractA methodological overview of proteome analysis is provided along with details of efforts to achieve high‐throughput screening (HTS) of protein samples derived from two‐dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700–1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)‐based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time‐of‐flight (MALDI‐TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI‐TOF‐mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene‐product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half‐life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene‐products on biochemical flux; the influence of the environment, cell‐cycle, stress and disease on gene‐products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene‐products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large‐scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two‐dimensional (2‐D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.
Additional approaches to DNA typing of skeletal remains: The search for “missing” persons killed during the last dictatorship in Argentina
Authors: Corach Daniel , Sala Andrea , Penacino Gustavo , Iannucci Nancy , Bernardi Patricia , Doretti Mercedes , Fondebrider Luis , Ginarte Anahi , Inchaurregui Alejandro , Somigliana Carlos , Turner Silvana , Hagelberg Erika 
Published: May 2005

AbstractDNA typing techniques are among the most advanced tools for human identification and can contribute to the identification of poorly preserved skeletal remains. Ten thousand people are thought to have been killed during the last dictatorship in Argentina (1976–1983) and there are few official records on the identity of the victims or the location of burials. A mass grave containing 340 skeletons was excavated using archeological methods. A small number of individuals was identified by traditional forensic methods and one family group by mitochondrial DNA (mtDNA) analysis. Due to the lack of antemortem physical information on many of the victims, the application of molecular methods is imperative to speed up the identification process. We have tested two molecular screening methods, Y chromosome‐specific short tandem repeats (DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393) and amplification of autosomal microsatellites using nested primers. These methods can complement solely matrilineal mtDNA sequence data in the identification of “missing” persons.