bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Rotofor in two‐dimensional electrophoretic analysis: Identification of a 100 kDa monoclonal IgG heavy chain in myeloma serum
Authors: Goldfarb Marcia F. 
Published: May 2005

AbstractAfter preparative nondenaturing isoelectric focusing in a Rotofor of 1 mL human serum which showed on two‐dimensional electrophoresis a monoclonal kappa light chain pI < 5.0, the monoclonal kappa light chain was identified in the tubes with pH 7.29 – pH 6.96, indicating that the chain was not free and was most likely part of an immunoglobulin molecule. The combined fractions of the Rotofor were concentrated back to 1 mL and aliquots were separated by two‐dimensional electrophoresis, followed by Western blotting. Enzyme immunoassay of the blot with A′ human IgG (γ chain specific) revealed a monoclonal heavy chain at a molecular size of approximately 100 kDa. Diffuse areas at 50 kDa (the attributed size of γ heavy chain), 100 kDa, and 150 kDa were stained also, but the strong, restricted (picket fence) bands indicating a monoclonal pattern were at 100 kDa. The monoclonal light chain appears to be part of a large or dimeric IgG molecule.
DNA shape and separation efficiency in polymer media: A computerized method based on electrophoretic mobility data
Authors: Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractThe computer program ELPHOFIT for evaluation of the nonlinear plots of log‐(mobility) vs. polymer concentration (Ferguson plots) in terms of molecular and polymer properties has been extended to yield a measure of the molecular sieving capacity of the polymer medium. The usefulness of the extended program, version 2.2, was exemplified by the evaluation of DNA shape and separation efficiency in solutions and gels of agarose and polyacrylamide, using previous reports in the literature as a data base. That application of the extended program yields the following results:(i) The size of migrating DNA can be compared with an equivalent sphere having the same free mobility for a particular set of experimental conditions. The decrease in size of the equivalent sphere with polymer concentration previously demonstrated for agarose solutions applies to all of these polymer media; it reveals a steep, hyperbolic decline of that radius in uncrosslinked polyacrylamide solutions in contrast to the shallow decline in the other three media. (ii) The separation efficiency of polyacrylamide gels exceeds that of uncrosslinked polyacrylamide solutions; the separation efficiency of agarose solutions for DNA smaller than 1 kb in length is higher than that of polyacrylamide solutions. Program ELPHOFIT 2.2 is available on request from the first author.
Retardation of serum albumin migration in native polyacrylamide gel electrophoresis by the incorporation of blue dextran in the gel matrix
Authors: Pemberton Alan D. , John Huw A. 
Published: May 2005

AbstractBlue dextran was incorporated into the stacking layer of one‐dimensional polyacrylamide gels to prevent the migration of albumin into the resolving gel during the electrophoresis of native plasma samples. Human and horse plasma proteins normally masked by albumin were revealed by this method.
Pulsed‐field polyacrylamide gel electrophoresis: Basic phenomena and applications
Authors: Ito Takashi , Hohjoh Hirohiko , Sakaki Yoshiyuki 
Published: May 2005

AbstractPulsed‐field gel electrophoresis (PFGE) using polyacrylamide gels, termed pulsed‐field polyacrylamide gel electrophoresis (PF‐PAGE), had been developed for the effective separation of linear DNAs from circular ones [1]. The first generation PF‐PAGE employed horizontal polyacrylamide gels run in a contour‐clamped homogeneous electric field (CHEF) apparatus. The second generation system, using a vertical slab gel in a discontinuous buffer system and field inversion gel electrophoresis (FIGE), was found to be easier to handle and requires a much shorter time for separation than the previous one [2]. In this report, basic aspects of the second generation PF‐PAGE, such as the effects of a discontinuous buffer system and field inversion on the DNA migration in polyacrylamide gels, were investigated. The results indicate that the periodic inversion of electric field can broaden the resolving capability of polyacrylamide gels, enabling DNAs that otherwise fail to enter polyacrylamide gels to be resolved in such systems. Successful and possible applications of PF‐PAGE techniques are also discussed.
The 0.7 to 3.3 megabase chromosomes from Candida, Kluyveromyces, and Pichia provide accurate size standards for pulsed field gel electrophoresis
Authors: Zhang Tai Yong , Fan Jian‐Bing , Ringquist Steven , Smith Cassandra L. , Cantor Charles R. 
Published: May 2005

AbstractPulsed field gel electrophoresis was used to size intact chromosomal DNAs from Candida albicans, Kluyveromyces lactis, Pichia scolyti, and Pichia mississippiensis by optimization methods using, as size standards, concatenated bacteriophage lambda DNA, and intact and NotI digestion products of Schizosaccharomyces pombe chromosomal DNAs. These newly sized fungal DNAs can now serve as convenient and accurate size standards for DNA molecules between 0.7 and 3.3 megabases (Mb). These size standards are valid over a wide range of different electrophoretic conditions.
Influence of the agarose matrix in pulsed‐field electrophoresis
Authors: Kirkpatrick Francis H. , Dumais Maxine M. , White Hugh W. , Guiseley Kenneth B. 
Published: May 2005

AbstractProperties of agarose potentially relevant to PFGE (pulsed‐field gel electrophoresis) are reviewed, and some new information is presented. Agarose polymers appear to have molecular weights in the range of 100 000 to 200 000 Da, but this is not tightly related to the effective gel strenght. Agarose has some residual charge, and hence exhibits electroendosmosis (EEO). It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strenght, non‐borate buffers. This increase is especially noticeable in the relatively long experiments required for separation of large DNAs. It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda‐phage restriction fragments in the same lane. Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables. Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE.
Trace analysis of proteins by capillary zone electrophoresis with on‐column transient isotachophoretic preconcentration
Authors: Foret František , Szökő Eva , Karger Barry L. 
Published: May 2005

AbstractThe qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co‐ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co‐ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface‐coated fused‐silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 μL was injected into a 75 μm ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10−7–10−8 M, with detection limits of approximately 10−9 M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.
Screening and characterization of biopharmaceuticals by high‐performance capillary electrophoresis with laserinduced native fluorescence detection
Authors: Lee Thomas T. , Lillard Sheri J. , Yeung Edward S. 
Published: May 2005

AbstractHigh‐performance capillary electrophoresis (HPCE) with laser‐induced native fluorescence (LIF) detection is used to address significant problems in the quality control of biopharmaceuticals. All of the biopharmaceuticals studied can be detected at subnanomolar levels with linear dynamic ranges of at least 3 orders of magnitude. HPCE/LIF can determine impurities in “purified” biopharmaceuticals present in amounts less than 0.01% (i.e., at 4 × 10−11 M) that of the major component. With HPCE/LIF, detection sensitivity is thus no longer a concern in the assaying of active ingredients in biopharmaceutical dosage formulations. The peptide mapping of biopharmaceuticals present at 1 × 10−7 M (or an injected limit of detection of 60 amol) is presented. Also, kinetic information on the reaction of a recombinant enzyme‐drug with its substrate present at the micromolar level has been extracted from electropherograms acquired in real‐time.
Contribution of secondary structure to DNA mobility in capillary gels
Authors: Konrad Kenneth D. , Pentoney Stephen L. 
Published: May 2005

AbstractIntramolecular base pairing at the 3′ end of single‐stranded DNA molecules can cause increased electrophoretic mobility. In DNA sequencing separations this can result in compressions, where multiple oligomers differing in length by one base comigrate, which complicate sequence analysis. Using a novel approach to the application of capillary electrophoresis to DNA sequencing, incorporation of formamide into capillary gels, different separation field strengths, and external heating of the capillary are examined for their ability to resolve compressions. The identity of the 3′ terminal nucleotide has also been observed to influence oligomer mobility in cross‐linked acrylamide slab gels. We utilize automated data collection from DNA sequencing separations to show that a similar effect is also present in linear acrylamide capillary gels run at high field strengths. We also demonstrate the ability of capillary electrophoresis to separate synthetic oligos of the same length but differing in composition by one base, or of the same base composition but different sequence.
On the pH dependence of polymerization efficiency, as investigated by capillary zone electrophoresis
Authors: Caglio Silvia , Righetti Pier Giorgio 
Published: May 2005

AbstractThe conversion efficiency of 5 different catalyst systems for polyacrylamide gel polymerization has been assessed as a function of pH of the gelling solution, in the pH 4.0–10.0 range. The system of persulfate‐N,N,N′,N′‐tetramethylethylene‐diamine (TEMED) gives optimal incorporation of monomers only in the pH 7.0–10.0 range; at progressively acidic pH values, the conversion drops markedly until, at pH 4.0, no gelation occurs. In the system of riboflavin‐TEMED, the opposite behavior is observed: good conversions in the pH 4.0–7.0 interval (with a peak at pH 6.2), followed by progressive worsening at alkaline pH values until, at pH 10.0, no gelation occurs. An excellent catalyst system appears to be the complex comprising methylene blue, toluene‐4‐sulfinic acid and diphenyliodonium chloride, which is able to guarantee > 95% conversion in the pH 4.0–8.0 range. Lower efficiencies are obtained at pH 9.0 and 10.0 (down to 81 % conversion) but gelation still occurs in the entire pH range. Two other systems (ascorbic acid, ferrous sulfate and hydrogen peroxide in one case, and persulfate, TEMED and hydrosul‐fite, in another case) have been investigated. The former has some efficiency only at pH 4 (81 % conversion), with a rapid drop as pH increases (only 48 % incorporation at pH 6). The latter exhibits the opposite behavior: 70% efficiency at pH 4.0, increasing to 92 % conversion at pH 6.0.
Determination of human alcohol dehydrogenase and acetaldehyde dehydrogenase genotypes by single strand conformation polymorphism in discontinuous buffer electrophoresis
Authors: Walzer Claude , Turler Hans , Balant Luc , Golaz Olivier , Hochstrasser Denis F. , Monteiro Maristela , von Wartburg Jean‐Pierre 
Published: May 2005

AbstractUnder appropriate conditions single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products allows the detection of single base mutations in a given DNA fragment. We adapted this method for the routine determination of allele variants of human alcohol and acetaldehyde dehydrogenase without radioisotopic labeling. After PCR amplification of the selected exon, the DNA fragments were heat‐denatured and loaded on a polyacrylamide gel containing glycerol. For electrophoresis a discontinuous buffer system was used with sulfate as leading ion and borate as trailing ion. The DNA bands were revealed by silver staining. Acrylamide concentrations, ionic strength and electrophoresis temperature were systematically investigated for each DNA fragment. The polymorphisms detected by SSCP were identical to those found by hybridization with 32P‐labeled allele‐specific oligonucleotides. This method avoids the use of radioactivity, is less expensive and simpler than the allele‐specific oligonucleotide (ASO) methodology and thus particularly suited for routine analysis.
Phenotypic change and altered protein expression in X‐ray and methylcholanthrene‐transformed C3H10T1/2 fibroblasts
Authors: He Chaoying , Merrick B. Alex , Witcher Lora L. , Patterson Rachel M. , Daluge Dennis R. , Selkirk James K. 
Published: May 2005

AbstractThe morphology, growth properties and cellular protein patterns from parent and two transformed C3H10T1/2 cell lines were analyzed to associated the phenotypic and protein differences with cell transformation. Transformed 10T1/2 cells were obtained by colony isolation after exposure of parent 10T1/2 cells to methylcholanthrene (MCA‐1 cell line) or X‐ray irradiation (XR‐III cell line). Compared to parent 10T1/2 and MCA‐1 cells, XR‐III cells were much smaller in size and exhibited the highest growth rate, greatest cell saturation density, increased plating efficiency and greater expression of proliferating cell nuclear antigen. MCA‐1 cells showed intermediate characteristics between parent and XR‐III cells. Among the three cell lines, only XR‐III cells showed anchorage‐independent growth in soft agar. When [35S]methionine‐labeled whole cell lysate proteins were separated by two‐dimensional polyacrylamide gel electrophoresis, computer comparison algorithms revealed a 97% similarity in protein profiles among almost 800 proteins detected from each cell line. However, comparison of proteins patterns of the transformed cell lines to that of parent 10T1/2 cells showed that 30 and 20 proteins were induced or repressed in XR‐III cells and MCA‐1 cells, respectively. Similarly, 81 and 24 proteins showed significant quantitative changes (threefold or greater) in XR‐III and MCA‐1 cells, respectively, as compared with parent 10T1/2 cell proteins. The ahchorage‐independent growth and increased proliferation properties of XR‐III cells suggest a later stage of transformation compared to MCA‐1 cells. Overall, these data suggest that gradational morphologic and phenotypic aberrancy in MCA‐1 and XR‐III cell lines from parent cells is accompanied by an increased diversity in protein expression in a limited set of proteins, some of which may play important roles in the multistep transformation process.
Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment
Authors: Ho Teh‐Yuan , Russo Jose , Russo Irma H. 
Published: May 2005

AbstractNumerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF‐10F is accompanied by the immunocytochemical expression of inhibin α and β subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2‐D PAGE in the immortalized HBEC MCF‐10F cells teated in vitro with 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF‐10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2‐D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded proteins in the molecular mass range of 24–72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparision with control cells. The hCG‐treated cells actively synthesized a 33‐kDa polypeptide which was not present in control cells. The nature of this hCG‐inducible 33 kDa protein elucidated by immunoprecipating [S35]methione‐labeled proteins with antisera directed against rat inhibin subunit α and βb. The immunoprecipitation revealed a 14 kDa protein band and an 18 kDa band whose syntheses were stimulated by hCG treatment, which corresponds to the inhibin α and βb subunits, respectively.
Chiral separations by capillary electrophoresis: Present state of the art
Authors: Vespalec Radim , Boček Petr 
Published: May 2005

AbstractCapillary electrophoresis is a powerful tool for chiral separation of ionogenic enantiomers in solutions. This article brings an overview of the theory of electrophoretic separations with special emphasis on enantiomer – chiral selector equilibria, followed by a survey of indirect separations, based on formation of diastereomers with different electrophoretic mobilities, and a comprehensive appraisal of direct separations when the chiral recognition stems from (i) hostguest interactions using cyclodextrins and crown ethers as hosting agents, (ii) ligand exchange mechanism, (iii) affinity interactions or (iv) a combination of solute – chiral selector interactions with micellar electrophoretic transport. Finally, some trends in chiral separations by capillary electrophoresis are discussed.
Micellar electrokinetic capillary chromatography of benzodiazepines in human urine
Authors: Schafroth Matthias , Thormann Wolfgang , Allemann Daniel 
Published: May 2005

AbstractThe determination of the major urinary compounds of eight common benzodiazepines, flunitrazepam, diazepam, midazolam, clonazepam, bromazepam, temazepam, oxazepam, and lorazepam, by micellar electrokinetic capillary chromatography (MECC) is shown to be a simple and attractive approach for confirmation testing of these drugs in human urine. After enzymatic hydrolysis and extraction using mixed‐mode solid‐phase cartridges and a two‐step elution protocol, fractions were analyzed in a phosphate/borate buffer (pH 9.3) containing 75 mM sodium dodecyl sulfate and small amounts of isopropanol, methanol and/or acetonitrile using an instrument with on‐ column multi‐wavelength detection. The presence of these compounds could unambiguously be confirmed in patient urines which tested positive for benzodiazepines using a commercial enzyme multiplied immunoassay screening technique (EMIT). The sensitivity of the MECC assay is demonstrated to be better than that of EMIT. MECC analysis of one patient urine which tested negatively employing EMIT revealed the presence of lorazepam, this demonstrating that false‐negative results from the initial immunological screening process can be recognized using MECC. For one example, 7‐aminoflunitra‐zepam, the MECC data are shown to agree well with those obtained by gas chromatography/mass spectrometry.
Chiral separation of basic drugs by capillary zone electrophoresis with cyclodextrin additives
Authors: Bechet Isabelle , Paques Philippe , Fillet Marianne , Hubert Philippe , Crommen Jacques 
Published: May 2005

AbstractThe enantiomers of a series of basic drugs were separated in capillary zone electrophoresis (CZE) in phosphate buffers, pH 3, containing β‐cyclodextrin or one of its derivatives as chiral selectors and uncoated fused silica capillaries thermostated at 15°C. The nature of the cationic component of the background electrolyte was found to have a significant influence on achiral resolution and peak symmetry. The best results were obtained with triethanolamine, which was then used to adjust the buffer pH in all further experiments. The effects on chiral resolution of the nature and concentration of cyclodextrin, of the addition of methanol, and of capillary temperature were studied. Maximum resolution was obtained at a particular cyclodextrin concentration for each analyte, depending on the affinity of the analyte for this cyclodextrin. On the basis of the results, the effects of methanol addition and temperature on enantiomeric resolution could be explained and predicted. Numerous chiral separations are presented and suggestions for the rapid optimization of CZE enantioseparations with cyclodextrin additives are given.
On the possibility of performing chiral wall‐coated open‐tubular electrochromatography in 50 μm internal diameter capillaries
Authors: Vindevogel Johan , Sandra Pat 
Published: May 2005

AbstractRecent results have demonstrated acceptable enantiomer separations in open, 50 μm internal diameter capillaries, coated with a chiral stationary phase, a technique known as electrochromatography. According to existing literature, smaller column diameters, 10 μm or less, are generally preferred for this mode of operation. The conditions under which the wider columns can be used are discussed.
Screening of a mouse/human Y‐chromosomal cosmid library for gene candidates and markers by using (short) oligonucleotide probes
Authors: Träger Thorsten , Schmidt Petra , Epplen Jörg T. 
Published: May 2005

AbstractA comprehensive approach is described for the identification of sequences of interest from a human Y chromosomal cosmid library via (short) consensus oligonucleotide probes. It involves the ordering of cosmid clones grown in microtiter plates onto small filter membranes by a robot workstation. A high number of the clones are characterized by their repetitive sequence content, either by ubiquitously interspersed simple tandem blocks or by Y‐specific elements. The Y chromosomal repeat (DYZ2) appears underrepresented in the library. In contrast many novel microsatellite marker systems can now be developed for the Y chromosome on the basis of the simple repeat blocks described here. Though novel genes were not yet delineated so far, a number of candidate sequences with high coding potential and other interesting characteristics are described.