bluuebunny/crossref_metadata_2025_split huggingface dataset:
Subtyping of alkylated human orosomucoid: Evidence for a duplicated gene, ORM1*F2S
Authors: Rocha Jorge , Amorim António , Luckenbach Christine , Kömpf Jost , Ritter Horst 
Published: May 2005

AbstractIsoelectric focusing of human orosomucoid (ORM) was studied following different sample treatment. It is shown that: (i) alkylation with iodoacetamide leads to a drastic change in the isoelectric point (pI) of both ORM1 F2 and ORM2 A gene products and greatly improves the discrimination between ORM1 F1 and ORM1 F2; (ii) previous reduction of the molecule with dithiothreitol partially inhibits the pI transitions with resultant artifactual ORM1 F1F2S patterns that correspond in most cases to F2S phenotypes. With the technique now described, the persistence of three ORM1 gene products was found in only one individual and the segregation analysis is consistent with the existence of a rare ORM1*F2S haplotype.
Two‐dimensional motion of DNA bands during 120° pulsed‐field gel electrophoresis
Authors: Neitzey Lynn M. , Hutson M. Shane , Holzwarth George 
Published: May 2005

AbstractThe position and velocity of a band of double‐stranded, linear DNA from bacteriophage G were measured during 120° pulsed‐field gel electrophoresis, using a video micrometer. Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction. For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model. However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity. This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations. The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this. The sharp velocity spikes are consistent with the idea that, for T> T*, a large fraction of the DNA chains are stretched into U‐shaped or herniated configurations. When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension. Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction.
Effect of one‐dimensional pulsed‐field gel electrophoresis on linear and circular DNA
Authors: Turmel Chantal , Noolandi Jaan 
Published: May 2005

AbstractA summary of the three main one‐dimensional pulsed‐field strategies (zero‐integrated field, forward‐biased field, and high frequency modulation) used for separating DNA molecules without band inversion within a preselected size range is given. Each of these strategies has size‐specific features which make separations up to 6 Mbp possible. We applied the same methodology to circular DNAs varying in size from 2 kbp to about 4 Mbp. The migration of intermediate‐sized circular plasmids (50 kbp–400 kbp) under these pulse conditions remains unexplained. On the other hand, preliminary results show that the migration of very large molecules, which are expected to be circular, comigrate with linear chromosomes of the same size under certain pulse conditions. We hypothesize that sample preparation, or the effect of the pulsed field, can create breakage and linearize very large circular DNAs, or that very large circular DNAs (> 2 Mbp) act like linear DNAs of the same size when submitted to one‐dimensional pulsed‐field gel electrophoresis conditions. The most likely possibility is that some of the circular DNAs have been linearized with one break during sample preparation, giving rise to a band at about 4 Mbp. The circular DNAs with more than one break may form an indistin‐guishable smear.
High‐performance capillary electrophoresis of glycoconjugates
Authors: Novotny Milos V. , Sudor Jan 
Published: May 2005

AbstractCapillary electrophoresis (CE) has recently emerged as a highly promising technique for the analysis and structural study of glycoconjugates. This article reviews the current status of CE and its potential in glycobiology. The separation media suitable for the analysis of monosaccharides, oligosaccharides and very large carbohydrate‐based biomolecules are discussed. The detection aspects emphasize chemical derivatization approaches to tagging the glycoconjugates of interest for enhanced response in absorption and fluorescence spectroscopy.
Capillary zone electrophoresis of biological substances with fused silica capillaries having zero or constant electroosmotic flow
Authors: Smith Joel T. , Rassi Ziad El 
Published: May 2005

AbstractA series of capillary surface modifications entailing multilayered coatings were introduced and evaluated in capillary zone electrophoresis of biological substances, e.g., proteins, peptides, oligosaccharides and nucleotides. In one set of surface modifications, large molecular weight hydroxypropyl cellulose afforded “zero” flow capillaries, which were used as precursors for developing anodal flow capillaries. When “zero” flow capillaries were further functionalized with a charged polyethyleneimine layer to which a top polyether layer was covalently attached, the resulting anodal electroosmotic flow was relatively weak due to the high viscosity of the coated wall imparted by the hydroxypropyl cellulose layer. Capillaries with relatively strong and constant anodal electroosmotic flow were best achieved when the inner capillary surface was first chemically derivatized with methylated (i.e., quaternarized) polyethyleneimine hydroxyethylated. This hydroxylated and permanently charged polymeric coating yielded constant anodal flow regardless of electrolyte pH. The hydroxyl groups of the charged polymeric coating permitted the covalent attachment of polyether chains, which minimized electrostatic interaction between the positive charges of the polymeric layer and oppositely charged biopolymers. Under these conditions, rapid transport of acidic biopolymers past the detection point in the separation capillary could be achieved, and relatively high plate counts were obtained.
Application of a diode‐array detector in capillary electrophoresis
Authors: Beck Wolfgang , van Hoek Roger , Engelhardt Heinz 
Published: May 2005

AbstractIn the last decade diode‐array detection has proved to be extremely useful in high performance liquid chromatography in recording UV‐visible spectra directly and on‐line in the column effluent. In capillary electrophoresis (CE) only fast‐scanning detectors with long scan times (up to 2 s) are commercially available. A home‐made CE instrument with a diode‐array detector (DAD) was built and compared with a commercial fast‐scanning detector. It is shown that the fast‐scanning detector is advantageous when less than eight wavelengths are monitored. Due to the high data collection rate the DAD is superior if high resolution UV‐visible spectra are acquired or if several spectra of a narrow peak (usual peak width in CE below 5 s) are needed for peak purity control. It is shown that DAD detection can help to develop separation methods, to identify peaks according to their UV‐visible spectra, and to check peak purity.
A three‐dimensional plot for the display of transverse pore gradient gel electrophoresis data: Application to a kinetoplast DNA fragment of planar circular conformation
Authors: Wheeler David , Chrambach Andreas 
Published: May 2005

AbstractA 219 bp fragment of kinetoplast DNA from Crithidia fasciculata has previously been shown to exhibit a gel concentration dependent, anomalously low migration rate in 3–10% polyacrylamide transverse pore gradient gels relative to that of straight standards. The anomaly was interpreted in accordance with previous results of Marini et al. [1] as being due to the planar circular conformation of the fragment. The transverse pore gradient gel pattern is displayed here as a three‐dimensional plot of migration distance vs. gel concentration and DNA length for the kinetoplast fragment and straight DNA standards. The plot yields a smooth surface for the standards clearly separated from the curve of the kinetoplast DNA fragment. Thus, transverse gradient gel electrophoresis in conjunction with the three‐dimensional plot provides a unique method for the recognition of abnormal conformational features of DNA on the basis of electrophoretic mobility in a pore‐size gradient.
An electrophoretic study of the thermal‐ and reductant‐dependent aggregation of the 27 kDa component of ammonia monooxygenase from Nitrosomonas europaea
Authors: Hyman Michael R. , Arp Daniel J. 
Published: May 2005

AbstractStandard protocols for sample preparation for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) typically involve the combined use of heat and a reductant to fully disrupt protein‐protein interactions and allow for constant ratios of SDS‐binding to individual polypeptides. However, 14C‐labeled forms of the membrane‐bound, active‐site‐containing 27 kDa polypeptide of ammonia monooxygenase from Nitrosomonas europaea undergo an aggregation reaction when cells or membranes are heated in the presence of SDS‐PAGE sample buffer. The aggregate produced after heating at 100°C is a soluble complex which fails to enter the stacking gel in discontinuous SDS‐PAGE gels. The extent of the aggregation reaction is dependent on the temperature of sample preparation, and the reaction exhibits first‐order kinetics at 65°C and 100°C (rates constants = 0.07 and 0.35 min−1, respectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of SDS does not significantly affect the rate of aggregation. The aggregated form of the 27 kDA polypeptide can be isolated by gel‐permeation chromatography in the presence of SDS. The aggregated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occurs with 14C2H2‐labeled polypeptides in other species of autotrophic nitrifiers and a methanotrophic bacterium which expresses the particulate form of methane monooxygenase. We conclude that strongly hydrophobic amino acid sequences present in ammonia monooxygenase are responsible for the aggregation phenomenon.
Detection of neurotensin in tissues by capillary zone electrophoresis
Authors: Denoroy Luc 
Published: May 2005

AbstractAn electrophoretic method for detecting neurotensin in tissues was developed. Homogenates of rat duodenum and adrenal glands were first extracted by solid‐phase extraction and purified by reversed‐phase high‐performance liquid chromatography. The neurotensin‐rich fraction was further analyzed by capillary zone electrophoresis (CZE) using a commercial instrument with UV detection. A minor peak was detected as neurotensin and its identity was further confirmed by performing several CZE analyses at different pH values. Such an approach could be used to analyze with good molecular specificity the neuropeptides in some human tissues.
Factors affecting the determination of drugs and endogenous low molecular mass compounds in human serum by micellar electrokinetic capillary chromatography with direct sample injection
Authors: Schmutz André , Thormann Wolfgang 
Published: May 2005

AbstractFactors influencing the establishment of an analytical window in front of the solubilized proteins in micellar electrokinetic capïllary chromatography (MECC) with direct serum injection (DSI) are discussed. Both drugs and endogenous low molecular mass compounds eluting within the analytical window are identified concurrently by multi‐wavelength absorption detection. Variables such as the concentration of the micelle forming substance, ionic strength, applied voltage, initial sample zone length, capillary length, selected buffer additives, insufficient renewal of the buffer in the anodic buffer vial and sample matrix are shown to impact MECC of endogenous compounds and model drugs, such as antiepileptics. For two drugs eluting within the analytical window, phenobarbital and ethosuximide, serum levels determined by DSI with external calibration are shown to compare well with levels obtained after liquid‐liquid extraction and internal calibration (use of an internal standard). In addition, reproducibility of both assays is excellent. The limit of employing DSI is demonstrated with the determination of the hydrophobic drug phenytoin. Using an automated, commercial instrument and naproxen as model drug, high‐speed MECC separations of high reproducibility and with a throughput of 12–15 samples per h are presented.
A theoretical approach to chiral capillary electrophoresis with some practical implications
Authors: Wren Stephen A. C. , Rowe Raymond C. , Payne Robin S. 
Published: May 2005

AbstractA simple model has been proposed which explains enantiomeric resolution in capillary electrophoresis in terms of differential interactions with a chiral selector and differential migration of the two enantiomer‐chiral selector complexes. The equations derived allow predictions to be made and there is good support for the model in literature data.
Enantiomer separation using mobile and immobile cyclodextrin derivatives with electromigration
Authors: Mayer Sabine , Schurig Volker 
Published: May 2005

AbstractThe principle of electrochromatography is applied to enantiomer separation with wall‐coated capillaries of 50μm (ID). As chiral stationary phase an immobilized polysiloxane containing chemically bonded monokis‐6‐O‐octamethylene‐permethyl‐cyclodextrin (β‐ or γ‐Chirasil‐Dex) was used. The results are compared with the conventional method of adding cyclodextrin derivatives as chiral pseudostationary phase to the running buffer.
Separation of enantiomers by affinity electrokinetic chromatography using avidin
Authors: Tanaka Yoshihide , Matsubara Norio , Terave Shigeru 
Published: May 2005

AbstractAvidin, a basic protein isolated from egg white, was employed as a chiral selector in affinity electrokinetic chromatography (EKC) for the separation of acidic enantiomers. Optical isomers of vanilmandelic acid, warfarin, ibuprofen, ketoprofen, flurbiprofen, and folinic acid were successfully resolved within 20 min with 25 μM avidin in a linear polyacrylamide‐coated capillary under weakly acidic conditions. The effects of pH, the concentration of avidin, addition of an organic solvent such as ethanol and 2‐propanol, and temperature on enantioselectivity were investigated. The choice of pH and the organic solvent additive was critical to improve the separation. Some general considerations about affinity EKC are also described, based on a simple one‐site interaction model.
Fluorescent‐based typing of the two short tandem repeat loci HUMTH01 and HUMACTBP2: Reproducibility of size measurements and genetic variation in the swedish population
Authors: Holgersson Stig , Karlsson Jan‐åke , Kihlgren Anne , Rosén Björn , Savolainen Peter , Gyllensten Ulf 
Published: May 2005

AbstractThe aim of this study was to investigate the reproducibility of genetic typing of two tetrameric short tandem repeat (STR) loci and the extent of genetic variation in the Swedish population. An automated, fluorescent‐based Applied Biosystems 373A sequencer was used for typing of the HUMTH01 and HUMACTBP2 loci (also named SE33). The former locus has seven alleles in the size range of 154–174 bp, while the latter is a complex locus with more than 32 alleles in the range of 227–316 bp. Using different fluorescent dyes, polymerase chain reaction (PCR) products from the two STR loci were sized in one lane using an internal size standard. In order to compare within‐ and between‐gel reproducibility of fragment size estimates, a control sample was typed three times on each of 20 gels. Within the gel, the standard deviation (SD) of fragment size variability was less than 0.1 bp for four fragment sizes between 158–291 bp. Standard deviations between gels were slightly higher for the two shorter fragment sizes (HUMTH01), while the larger fragments varied between 0.3 and 0.4 bp (HUMACTBP2). The amount of genetic variation was investigated in samples from three Swedish cities (n = 301). Seven alleles were found at HUMTH01 and the observed heterozygosity was 0.77. At the HUMACTBP2 locus more than thirty alleles were found and the observed heterozygosity was 0.96. The observed genotype frequencies at HUMTH01 and HUMACTBP2 did not deviate significantly from Hardy‐Weinberg expectations. No indication of a significant excess of homozygotes was found at any of the loci. We conclude that both HUMTH01 and HUMACTBP2 can be reliably typed using the method described. However, the latter locus requires an allelic ladder to be run on each gel.
The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2‐aminoacridone
Authors: Jackson Peter , Pluskal Malcolm G. , Skea William 
Published: May 2005

AbstractA method is described for the electrophoretic analysis of fluorophore‐labeled acidic glycans. Various glycoproteins were treated with the enzyme, peptide N‐glycosidase F, to release mixtures of asparagine‐linked glycans that were then labeled with the fluorophore 2‐aminoacridone, and the resulting derivatives electrophoresed in high‐density polyacrylamide gels. The acidic glycans were separated with high resolution and distinct, well‐resolved fluorescent band patterns were produced. Neutral saccharides did not electrophorese in the system. Information on the structure of the glycans was revealed by the changes in the band patterns observed after the mixtures of the glycan fluorophore derivatives were treated with either of the enzymes neuraminidase or β‐galactosidase. Quantities of glycoprotein as little as 10 μg were analysed without difficulty. The electrofluorograms were viewed using a digital imaging system based on a cooled charge‐coupled device. The method was also demonstrated using purified acidic glycans of known structure. The method was easy to use, enabled the sensitive analysis of multiple samples in parallel and should be a useful addition to the range of glycan analytical techniques.
Preparative isoelectric focusing in multicompartment electrolyzers: Novel, hydrolytically stable and hydrophilic isoelectric membranes
Authors: Chiari Marcella , Nesi Marina , Roncada Paola , Righetti Pier Giorgio 
Published: May 2005

AbstractPreparative isoelectric focusing in multicompartment electrolyzers is based on the production of isoelectric membranes of precise isoelectric point, able to buffer at their pI value and to titrate proteins tangent to or crossing the membranes. Up to the present, such membranes have been based on polyacrylamide chemistry; acrylamide, however, is neither stable in acidic nor basic environments. We describe here novel membranes, produced with a unique monomer, N‐acryloylaminoethoxyethanol (AAEE). Poly(AAEE) membranes are extremely stable to alkaline hydrolysis (500 times more stable than polyacrylamide) and even more hydrophilic than the latter matrix. This allows production of highly reproducible membranes (these do not change their pI with time, since no acrylic acid is produced by hydrolysis upon storage) which do not adsorb proteins by hydrophobic interaction.
Capabilities and potentialities of transverse pore gradient gel electrophoresis
Authors: Chrambach Andreas , Wheeler David L. 
Published: May 2005

AbstractTransverse pore gradient gel electrophoresis is important as a tool for obtaining nonlinear Ferguson plots [log(mobility) vs. gel concentration], e.g. in application to DNA in polyacrylamide gels or to agarose gels, with the purpose of evaluating molecular properties (size, conformation, malleability) and gel fiber properties (fiber radius and length per unit volume). To date, it is capable of (i) yielding gel patterns (“Ferguson curves”) of migration distance vs. predicted % T‐range of the pore gradient, assuming its linearity;(ii) yielding information regarding molecular conformation from the intersection of Ferguson curves of unknowns (e.g. bent DNA) with those of standards; (iii) acquisition of Ferguson curves by computer, using prototype instrumentation; (iv) mathematical manipulation of acquired Ferguson curves to yielding Ferguson plots, providing that mobility in free solution has been assessed by capillary zone electrophoresis. The potentialities of the method remain unfulfilled to date due to (i) the unavailability, with a single exception, of an accurate and precise way to produce pore gradients of known shape; (ii) unavailability of a routinely applicable analysis for % T; (iii) unvailability of optimized, user‐friendly and foolproof instrumentation for computer acquisition of Ferguson curves, including the present inapplicability of a commercially available electrophoresis apparatus with intermittent optical detection to transverse pore gradient gels; and (iv) unresolved problems in the statistical evaluation of Ferguson curves.
Electrophoretic analysis of seed storage proteins from gymnosperms
Authors: Allona Isabel , Collada Carmen , Casado Rosa , Aragoncillo Cipriano 
Published: May 2005

AbstractSodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), pore gradient gel electrophoresis (PGGE) followed by SDS‐PAGE and Western blot analysis were used to characterize the seed storage proteins from seven gymnosperm species from the families Pinaceae (Abies alba, Cedrus atlantica and Picea abies), Cupressaceae (Biota orientalis, Chamaecyparis lawsoniana and Cupressus arizonica), and Taxaceae (Taxus baccata). SDS‐PAGE and PGGE X SDS‐PAGE indicate the presence of proteins with characteristics similar to the 7S globulins in all the species studied. Antibodies to a 7S globulin subunit from Pinus pinaster cross‐reacted with homologous polypeptides from Pinaceae species, but not with corresponding subunits from species belonging to other families. Also detected in each of the studied species, with the exception of A. alba and T. baccata, were proteins potentially homologous with 11S proteins. Of these proteins, only those of C. atlantica may be extracted by saline buffer, while the remainder require a dissociating agent. Antibodies raised against the small subunit from P. pinaster 11S protein recognized only the corresponding polypeptides from Pinaceae species. Overall, these results help clarify our knowledge of gymnosperm seed storage proteins.
Differential splicing of the glycophorin A mRNA
Authors: Omi Toshinori , Kajii Eiji , Iwamoto Sadahiko , Tsuchida Shuichi , Ikemoto Shigenori 
Published: May 2005

AbstractTwo truncated mRNAs, in contrast to the full length of mRNA associated with the glycophorin A gene (GpA‐TI, GpA‐TII), were isolated from erythroid cells cultured by the selective two‐phase liquid culture system for erythroid progenitors in peripheral blood from a normal individual. The GpA‐TI mRNAs displayed a direct transition from exon I to exon III, so that the deletion of exon II resulted in the deletion of 33 amino acids encoded by this exon. Furthermore, the GpA‐TII showed two direct transitions from exon I to exon III and from exon III to the exon V of the GpA gene. This mRNA lacked both exons II and VI, resulting in the deletion of 46 amino acids. Is is concluded that these truncated mRNAs are transcribed from the same gene as the GpA gene and correspond to splicing isoforms lacking different exons.