bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein alterations associated with gene amplification in cultured human and rodent cells
Authors: Conner Elizabeth A. , Wirth Peter J. 
Published: May 2005

AbstractGenomic stability was investigated in Chinese hamster ovary (CHO) and human hepatocellular carcinoma HepG2 cells selected for growth in the presence of cytotoxic concentrations of N‐(phosphonacetyl‐L‐asparate) (PALA). In CHO cells selected with 9 × LD50 PALA the carbamyl p‐synthetaase, asparate transcarbamylase and dihydroorotase (CAD) gene complex was amplified two‐fold while in HepG2 cells selected at comparable PALA concentrations a 7‐ to 10‐fold increase in the CAD gene was observed. Concomitant with amplification of the CAD gene were increases in CAD mRNA and protein expression in both CHO and HepG2 cells. In long‐term cultures of HepG2 cells the CAD gene underwent spontaneous amplification (5‐fold) in the absence of PALA treatment with increasing passage number. In an attempt to define proteins and/or family of proteins that may either directly or indirectly influence DNA amplification potential through a mechanism of enhanced genomic instability, immobilized pH gradient‐two‐dimensional polyacrylamide gel electrophoresis (IPG 2‐D PAGE) analysis of silver‐stained nuclear cytoplasmic polypeptides comcomitant with PALA resistance and CAD amplification was performed. Analysis of silver‐stained polypeptides from 3 × LD50 PALA‐selected CHO and HepG2 cells revealed no significant alterations in polypeptide expression. In CHO cells selected at 5 × and 7 × PALA LD50, and HepG2 cells selected at 5 × and 9 × PALA LD50, one subset of 4–8 polypeptides (pl: pI 7.2–7.6/36–38 kDa) were increased 2‐ to 3‐fold in both 5 × and 7 ×‐ and 5 × and 9 × LD50 PALA‐selected CHO and HepG2, respectively, while five relatively neutral‐to‐basic, low Mr polypeptides (p2: 18/7.30; p3: 16/7.00; p4: 14/7.00; p5: 14/7.40; and p6: 13.5/7.00) were markedly increased in CHO cells selected at 7 × LD50 PALA. In addition to these PALA‐associated increases, four polypeptides (p7a: pI 6.50/40 kDa; p7b: 6.55/40; p7c: 6.60/40: and p7d: 6.65/40) were significantly increased in high‐passage (p159) HepG2 cells undergoing spontaneous CAD gene amplification in the absence of PALA exposure. In CHO cells, polypeptides p7 a, b, d were increased while the expression of p7c (pI 6.60/40 kDa) was unaltered in 7 × LD50‐treated CHO cells. Although neither the identity nor biological function of polypeptides 1–7 is known, a proposed mechanism involving interaction with certain growth regulatory proteins such as p53 for mediating genomic instability is given.
Immune response to different doses of a hapten of fluorescein isothiocyanate analyzed by two‐dimensional affinity electrophoresis
Authors: Wang Pei , Nakamura Kazuyuki , Mimura Yusuke , Takeo Kazusuke , Tanaka Tatehiko , Fujimoto Masanori 
Published: May 2005

AbstractThe immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two‐dimensional affinity electrophoresis (2‐D AEP). The mice were immunized with different doses (0.5 μg, 5 μg, 50 μg, 500 μg and 2.5 mg) of FITC‐conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots. showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC‐BSA dosage. With a low dose (5 μg) most of the families showed high affinity (Kd < 1 μM). When the dose was increased, not only high affinity antibodies but also intermediate (1 μM < Kd < 50 μM) and low affinity (Kd > 50 μM) antibodies were produced. The increase of FITC‐BSA up to 500 μg markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC‐BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti‐FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgGl, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgGl, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2 and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.
Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single‐strand conformation analysis in mutation detection enhancement gels
Authors: Alonso Antonio , Martin Pablo , Albarran Cristina , Garcia Oscar , Sancho Manuel 
Published: May 2005

AbstractThe article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single‐strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty‐five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.
Dynamics of protein isoelectric focusing in immobilized pH gradient gels
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio 
Published: May 2005

AbstractThe protein migration velocity profile through the pH gradient in immobilized pH gradient gels (IPG) was investigated. Strong nonuniformities of migration velocity distribution, which could not be remedied by adding carrier ampholytes, were observed at the initial stages of a run (up to several thousand volthour products for wide pH gradients). The final (after a prerun) velocity distribution in IPG gels differs from the one containing carrier ampholytes, with maximum velocity being achieved not at the pH extremes but closer to neutrality. In some uniform pH media, created by either a single or by several Immobilines, essentially a nonconstant migration profile was also obtained immediately after a prerun. After a prerun, followed by a pause in the absence of current, the phenomenon of fast evolution of the final velocity profile was found.
Characterization of competitive binding, fluorescent drug immunoassays based on micellar electrokinetic capillary chromatography
Authors: Steinmann Lone , Thormann Wolfgang 
Published: May 2005

AbstractThis paper characterizes competitive binding, electrokinetic capillary‐based immunoassays for various drugs in human serum using reagents which were commercialized for fluorescence polarization immunoassays. After incubation of serum with the reactants, a small aliquot of the mixture is applied onto a fused‐silica capillary and tracers (fluorescein‐labeled drugs) and the antibodytracer complexes are separated and analyzed by micellar electrokinetic capillary chromatography with on‐column laser‐induced fluorescence detection. Examples studied include serum assays for theophylline, ethosuximide, paracetamol, salicylate and quinidine. With these assays, concentration‐dependent peaks produced by the free tracers or the antibody‐tracer complexes serve as the basis for quantitation. The sizes of the peaks produced are shown to be dependent on the applied power and the proportions of the reactants and serum employed. The separation medium permits effective characterization of tracers and antibody selectivities. Based on the high selectivity of the antibodies employed, the feasibility of the simultaneous performance of different immunoassays is demonstrated. For capillaries of 50 μm internal diameter (ID), separations are best performed at electric fields < 500 V/cm, this resulting in electrokinetic analyses within 4 to 10 min (capillaries of 20 to 50 cm effective length).
Some examples of the analysis of complex samples by micellar electrokinetic capillary chromatography with photodiode array detection
Authors: Vandepeer Jasmine , Trenerry V. Craige , Keogh Geraldine 
Published: May 2005

AbstractThe use of micellar electrokinetic capillary chromatography with photodiode array detection to assist in identifying the minor components in crude morphine, poppy straw and opium preparations, for the analysis of synthetic colours and other food additives in cordials and confectionery and for the identification of the ring substituted amphetamine derivative methylenedioxyethylamphetamine in illicit drug samples is demonstrated.
Dispersion coefficients of λ DNA in agarose gels during electrophoresis by fringe recovery after photobleaching
Authors: Tinland Bernard 
Published: May 2005

AbstractBy combining an electrophoretic cell with a fringe recovery after photobleaching setup, we have measured both the electrophoretic mobility μ and the dispersion coefficient D of double‐stranded λ‐DNA in agarose gel as a function of the applied electric field E. Besides the determination of the mobilities, experimental values of the exponential decay of the signal gives, for the first time, experimental values of the longitudinal Dx and transverse Dy dispersion coefficients (field E perpendicular and parallel to the light fringes, respectively). Time dispersion measurements are complicated by the existence of a bleached area drift time. We present an analysis of their different contributions. Both dispersion coefficients increase linearly with increasing field E and, in the range of field we used, Dx is larger than Dy. Our results are consistent with the recent throretical predictions of Slater (Electrophoresis 1993, 14, 1–7) and Duke et al. (Biopolymers 1994, 94, 239–247).
The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis
Authors: Sigmon Janie , Larcom Lyndon L. 
Published: May 2005

AbstractEthidium bromide (EtBr) is sometimes added to running buffer during the separation of DNA fragments by agarose gel electrophoresis. It is used because upon binding of the molecule to the DNA and illumination with a UV light source, the DNA banding pattern can be visualized. The mode of binding of EtBr is intercalation between base pairs. This binding changes the charge, weight, conformation, and flexibility of the DNA molecule. Since DNA molecules are sized by their relative movement through a gel compared to a molecular weight standard, mobility measurements can be critical to size determinations. After running two identical gels, one without EtBr and one with 0.25, 0.5, 0.75 or 1.0 μg/mL EtBr in the running buffer, the mobilities of λ HindIII DNA fragments were compared. The mobility of DNA was always less in the gels with EtBr. Using the reptation theory equation, which describes the mobility of DNA molecules through a gel, changes in frictional coefficients were calculated. It was determined that the change in frictional coefficients brought about by the addition of EtBr is directly proportional to the fraction of base pairs of a fragment bound to EtBr. This change in friction is greatest in the largest fragments, which suggests that the stiffening of the molecule by the EtBr binding is the cause for the decreased mobility.
Evaluation of the ALF DNA sequencer for high‐speed sizing of short tandem repeat alleles
Authors: Decorte Ronny , Cassiman Jean‐Jacques 
Published: May 2005

AbstractDNA typing of short tandem repeaat (STR) loci with automated real‐time analysis of the fluorescent polymerase chain reaction (PCR) products has given forensic DNA analysis a new dimension. In the present work, the ALF DNA sequencer was evaluated for automated size determination of tetra‐nucleotide STRs at high speed. Short gel plates, with a well‐to‐laser distance of 10 cm, allowed for the analysis of four STR loci (HUMvWF, HUMTH01, D21S11 and HPRT) in one gel lane in less than 75 min. Allele size determination was done with two external allelic ladders for each locus. Lane‐to‐lane variations were overcome by the inclusion in each lane of two fluorescent PCR products of constant size (123 and 375 bp) that migrated below and above the multiplex of the four STR loci. The accuracy of sizing and allele detection within and between different gels was high (99.89%) for all four STR systems investigated and the gels could be reloaded without a decrease in accuracy of the allele size estimation. This way, the throughput of the system was increased, which is of interest for linkage studies, gene mapping and population diversity studies.
Validation of the applied biosystems Prism™ 377 automated sequencer for forensic short tandem repeat analysis
Authors: Frazier Rachael R. E. , Millican Emma S. , Watson Stephanie K. , Oldroyd Nicola J. , Sparkes Rebecca L. , Taylor Karen M. , Panchal Sasikala , Bark Linda , Kimpton Colin P. , Gill Peter D. 
Published: May 2005

AbstractThe Applied Biosystems (ABI) PrismTM 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well‐to‐read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51°C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software Complete resolution of 1 base difference was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2‐fold higher than the 373A and the sensitivity was increased by one third.
Multilocus fingerprinting and single locus analyses in the great tit for paternity determination
Authors: Lubjuhn Thomas , Epplen Cornelia , Epplen Jörg T. 
Published: May 2005

AbstractIn order to arrive at detailed information about the occurence of alternative reproductive tactics in the great tit (Parus major) we examined parental lineages by multilocus fingerprinting in 51 great tit broods containing 429 nest‐lings. In 45% of the broods at least one nestling was found to stem from copulations with males other than the social mate of the female. Single broods contained up to 62.5% extra‐pair young (EPY). Single locus profiling revealed how many fathers were involved in extra‐pair paternity (EPP) within each brood: In most cases a single male is the genetic father of all EPY in a given brood.
Quick and easy molecular weight determination with Macintosh computers and public domain image analysis software
Authors: Seebacher Thomas , Bade Ernesto G. 
Published: May 2005

AbstractThe program “molecular weights” allows a fast and easy estimation of molecular weights (Mr), isoelectric point (pI) values and band intensities directly from scanned, polyacrylamide gels, two‐dimensional protein patterns and DNA gel images. The image coordinates of Mr and pI reference standards enable the program to calculate Mr and pI values in a real time manner for any cursor position. The program requires NIH‐Image for Macintosh computers and includes automatic band detection coupled with a densitometric evaluation.
Channel thickness variations could degrade resolution in electrophoresis
Authors: Holzwarth George 
Published: May 2005

AbstractVariations in the lateral dimensions of capillaries and ultrathin gels along their length could lead to a distortion of the sample zone because charged particles traveling near the wall must travel a longer distance under a smaller field than particles traveling along the centerline. The size of the effect is computed by solving Laplace's equation for the potential everywhere in a conducting slab with a discontinuity in thelateral dimensions and integrating the travel time along different streamlines.
Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light‐harvesting complex (LHC II)
Authors: Zolla Lello , Bianchetti Maria , Timperio Anna Maria , Mugnozza Giuseppe Scarascia , Corradini Danilo 
Published: May 2005

AbstractThe electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light‐harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0–10 with running electrolyte solutions containing either anionic, zwitterionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n‐octyl β‐D‐glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resoltuion of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.
The determination of choline in vitamin preparations, infant formula and selected foods by capillary zone electrophoresis with indirect ultraviolet detection
Authors: Carter Nicole , Trenerry V. Craige 
Published: May 2005

AbstractA method for the determination of choline in vitamin preparations, infant formula and selected foods by capillary zone electrophoresis with indirect ultraviolet detection at 214 nm is described. The “free” choline in the vitamin preparations was simply extracted with water, the solutions filtered and analysed. The “free” and “bound” choline in the other products was extracted with methanol. The near dry extract was then treated with hot saturated aqueous calcium hydroxide to hydrolyse the “bound” choline before analysis. A 75 cm × 75 μm uncoated fused silica capillary column with an electrolyte consisting of 5 mM 1‐methylimidazole, pH 4.5, and an operating voltage of 30 kV was used for all of the determinations. Using these conditions, choline migrates in less than 3 min and is well separated from the other components in the electropherograms. The tetramethylammonium ion was used as the internal standard. The instrument repeatability data for area calculation and migration time variation were good (coefficient of variation, CV, area calculation 1.4%, n = 10; CV for migration time variation 1.0%, n = 20). The level of detection for choline using this procedure is 5 mg/100 g.
Analysis of the biotin‐binding protein actinavidin using affinity capillary electrophoresis
Authors: Okun Vadim M. , Bilitewski Ursula 
Published: May 2005

AbstractAffinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand‐receptor interaction between the microbial biotin‐binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio‐ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio‐ON, permitting the evaluation of the biotin‐binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity inteaction, actinavidin in the sample decreases the amount of bio‐ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid‐phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.
The interface of capillary electrophoresis with high performance fourier transform ion cyclotron resonance mass spectrometry for biomolecule characterization
Authors: Severs Joanne C. , Hofstadler Steven A. , Zhao Zack , Senh Robert T. , Smith Richard D. 
Published: May 2005

AbstractThe interfacing of capillary electrophoresis (CE) with Fourier transform ion cyclotron resonance‐mass spectrometry (FTICR‐MS) and the factors which dictate obtainable performance (i.e., sensitivity, mass resolution, scan rate and duty cycle) are described. We demonstrate the current status of the technique with examples of capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) with FTICR analyses of proteins and oligonucleotides, and describe current limitations on sensitivity and scan speed. The first on‐line interfacing of capillary isoelectric focusing (CIEF) with FTICR is also demonstrated and shown to be effective for separating minor components of protein mixtures for on‐line mass spectral analysis. Finally, the potential for greatly improved performance based upon recent advances in FTICR instrumentation and methods is briefly described.
Capillary zone electrophoresis of inorganic anions with conductivity detection
Authors: Kaniansky Dušan , Zelenská Viera , Baluchová Daniela 
Published: May 2005

AbstractA carrier electrolyte system for capillary zone electrophoresis (CZE) resolving chloride, bromide, iodide, fluoride, nitrite, nitrate, sulfate, and phosphate in a hydrodynamically closed separation compartment is described. The carrier electrolyte combines the effects of pH, polyvinylpyrrolidone (PVP) and the counterionic constituent on the effective mobilities of the anions. In 300 μm ID capillary tubes made of fluorinated ethylene‐propylene copolymer (FEP), and with detection of analytes with the aid of an alternating current conductivity detector, detection limits in the range of 3–10 ppb could be achieved for 200 nL sample volumes. The separation efficiencies were in the range of 1.5–2.5 × 105 theoretical plates per meter for an adequate sample load. The reproducibility was evaluated for two concentration levels. For concentrations close to the limits of quantitation (50–120 ppb), the RSD values ranged from 1.5–12.6%, with the highest scatter for fluoride and phosphate. The RSD values were in the range of 0.4–1.5% for 300–1200 ppb concentrations of the anions. Typical analysis times were 2–5 min, depending on the anion species. A series of water samples (drinking, river, rain) was used to assess the practical applicability of the CZE method. The method is a suitable alternative for the determination of both anionic macro‐ and microconstituents in water with a good overall selectivity.