bluuebunny/crossref_metadata_2025_split huggingface dataset:
Global analysis of lymphocyte gene expression: Perturbation of H‐9 cells by infection with distinct isolates of human immunodeficiency virus – an exposition by multivariate analysis of a host‐parasite interface
Authors: Kettman John R. , Robinson Robin A. , Kuhn Lotte , Lefkovits Ivan 
Published: May 2005

AbstractAIDS is a progressive disease associated with steady loss of helper T cells and several other functions. As the disease evolves, cytopathogenic human immunodeficiency (HIV) variants of increasing virulence can be isolated from the host. The HIV is an unusually variable genome by virtue of a low replication fidelity. In this report we describe our effort to test the hypothesis that there is a correlation between virus variability and cytopathogenicity, and further, that there is an “impact” of the virus infection on the expression of host cellular genes. To search for such a relationship, we infected H‐9 cells (human CD4+ lymphoblastoid cell line) with each of 5 isolates of HIV of distinct origin and cytopathogenicity. To measure the influence of the virus infection on the expression of host cellular genes, shortly after infection, (3 h or 13 h), cells were radiolabeled and the radioactive polypeptides separated by two‐dimensional gel electrophoresis. Radiofluorographs were prepared and analyzed to determine relative rates of biosynthesis of cellular polypeptides. To organize the large amounts of data found, cluster analysis and principal component analysis were used to expose the data in formats that allowed a model construction. The rates of biosynthesis of many cellular polypeptides were altered upon viral infection in terms of both enhancements and impairment of biosynthesis. Some of the variation in polypeptide synthesis was isolate‐specific, while most alterations were of modest magnitude. There appears to be no “overall effect” associated with infection by a cytopathic variant of the virus. Polypeptides affected by the cytopathic variants were determined as targets for further investigation. The method used promotes the measurement of “ensemble” information that is characteristic of the process and it promotes the creation of models of virus action.
Electrophoretic and computer analysis of skeletal muscle used for cardiac assistance
Authors: O'Brien Geraldine A. , Cumming Debbie V. E. , Pattison Charles W. , Corbett Joseph M. , Dunn Michael J. , Yacoub Magdi H. 
Published: May 2005

AbstractSkeletal muscle has an inherent plasticity which allows it to undergo fibre type transformation when induced by a specific stimulus. Electrical stimulation has been used here to induce transformation of a predominantly fast type skeletal muscle towards a slow, more fatigue‐resistant phenotype, which is more suitable for use in long‐term cardiac assistance. Muscle samples from animals electrically stimulated for periods up to 6 months have been analysed by electrophoresis for myosin heavy chain (MHC) and myosin light chain (MLC) fast and slow isoforms. Densitometry and computer analysis have been used to determine the pattern of transformation of the different myosin subunits over this time period. MHC and MLC 2 fast to slow isoform switching preceded that of the alkali light chains (MLC1 and MLC3). After 3 months of stimulation the MHC slow isoform was found to have doubled in concentration relative to the unstimulated control muscle and by 4 months accounted for almost 50 % of the total MW content. The slow isoform accounted for 75 % of the MLC 2 after 4 months of stimulation. The protein products of mRNA isolated from stimulated muscle samples, translated in vitro and separated by electrophoresis, showed that transformation at the mRNA level preceded that at the protein level. By 2–4 weeks of stimulation MLC2 slow isoform mRNA represented over 60 % of the total MLC2 mRNA population. An understanding of the molecular structure of muscle during transformation provides insight into its haemodynamic performance in cardiac assistance.
An estimation of the sensitivity of in vitro translation using two‐dimensional gel analysis
Authors: Perrett Christopher W. , Whatley Stephen A. 
Published: May 2005

AbstractPoly (A+ mRNA species, isolated from 100‐day‐old rat brain, were analysed by in vitro translation and two‐dimensional gel electrophoresis. The synthesis of selected protein species was compared to actin on the basis of [35S]methionine incorporation. The estimated molar abundance of translation products varied from abundant species at 0.78% of the total to several are species, detectable below the 0.02% level. If these synthesis rates reflect the abundance of particular mRNAs in the mixture, this sensitivity limit compares well with accepted values using differential cDNA screening techniques. This analysis provides evidence that in vitro translation methodology is able to detect rarer mRNA species than is usually expected – these include similar abundance classes to library screening procedures.
The electrophoretic properties of a DNA cube and its substructure catenanes
Authors: Chen Junghuei , Seeman Nadrian C. 
Published: May 2005

AbstractElectrophoretic properties have been measured for a DNA molecule whose helix axes have the connectivity of a cube. This molecule is a topologically bonded complex of six cyclic 80‐mer molecules, in which each cycle corresponds to a face of the cube. Each cyclic molecule is doubly catenated to each of its four neighbors. Substructures of this molecule include a 5‐cycle structure lacking one strand, two topoisomers of 4‐cycle structures and two topoisomers of 3‐cycle structures. One 4‐cycle structure is a cyclic belt around the cube, lacking a top and a bottom, whereas the other lacks two catenated strands, such as the top and the front. One 3‐cycle structure is a linear belt of three cycles, and the other corresponds to the three cycles that surround a corner. Each of these molecules is separable from the others under appropriate gel conditions. We have measured mobilities and calculated Ferguson plots for each of these molecules on polyacrylamide gels under both native and denaturing conditions. The measurements have been made with 1.25, 2.5, and 5% crosslinking of the gels. The data show that the higher‐symmetry 3‐cycle and 4‐cycle structures migrate more slowly than their lower‐symmetry isomers, under conditions where their Ferguson plots are parallel.
Electrophoretic size separations in liquified agarose of polystyrene particles and circular DNA
Authors: Boček Petr , Chrambach Andreas 
Published: May 2005

AbstractPolystyrene sulfate particles of 0.37 to 1.78 μ in diameter are retarded in their electrophoretic migration in proportion to the concentration of agarose liquified above its gelling temperature. In the concentration range of 0.02 to 0.2% liquified agarose, the degree of this retardation in electrophoresis at 40°C is inversely related to particle size. By contrast, mitochondrial DNA (16 kb), plasmid pBR322 DNA (4 kb) and plasmid PSA509 DNA (3 kb) exhibit under the same conditions a degree of retardation which is proportional to their size. This confirms the existence of two divergent mechanisms of size separation similarly observed in other liquid polymer media, i.e. one based on collisions with the gel fiber (molecular sieving) and one based on exclusion from the fiber network (the electrophoretic equivalent of gel permeation).
A two‐dimensional gel database of human plasma proteins
Authors: Anderson N. Leigh , Anderson Norman G. 
Published: May 2005

AbstractAn updated two‐dimensional electrophoretic map of human plasma proteins is presented, together with a complete listing of the individual protein spots, their locations, size and isoelectric points relative to internal charge standards. Forty‐nine polypeptide species are identified, many consisting of multiple spots differing in glycosylation or sequence (e.g., immunoglobulins). A further series of 35 as yet uncharacterized proteins is indicated.
The gene‐protein database of Escherichia coli: Edition 4
Authors: VanBogelen Ruth A. , Neidhardt Frederick C. 
Published: May 2005

AbstractThe gene‐protein database of Escherichia coli has as its core an index that links each of the protein spots from a two‐dimensional polyacrylamide gel to the gene that encodes the protein. Additional information about each protein and its gene is generated from two‐dimensional gel analysis or collated from the literature to form the database. Earlier editions of the database have provided periodic updates of information. The current edition does this, but also introduces a new reference gel image produced by an electrophoresis system recently adopted in this laboratory. The new gel system was chosen because it offers an improved opportunity for other investigators to produce close replicas of the reference gel pattern, thereby allowing easier access to the information of the database and encouraging independent contribution to the database. The new gel format also is larger and hence more compatible with computer assisted image analysis, which has become essential for a project of this magnitude. This edition continues the use of the former reference gel images, but adds a reference image of an equilibrium gel of E. coli strain W3110 produced by the new standardized gel system. At this time, 55% of the protein spots annotated on the previous equilibrium reference gel for this organism have been located on the new reference image, and these identifications are included in the tables of the database.
Electrophoretic human leukocyte antigen HLA‐DQA1 DNA typing after polymerase chain reaction amplification
Authors: Barros Francisco , Carracedo Angel , Lareu Maria Victoria , Rodriguez‐Calvo Maria Sol 
Published: May 2005

AbstractAn electrophoretic method (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) is described which permits the identification of human leukocyte antigen HLA‐DQA1 types and subtypes without using allele‐specific oligonucleotide prodes and dot‐blot methodology. The procedure can be used in miniaturized gels in combination with automated electrophoretic systems. The PhastSystem is particularly recommended since temperature contol is essential. HLA‐DQA1*1 and DQA1*0301 can be distinguished in homoduplexes and DQA1*01 subtypes, DQA1*201 and DQA1*0401 in heteroduplexes (in only 5 including DNA extraction and PCR amplification). Additional variations to those recognized using commercially available dot‐blot methods can be provided since this this procedure permits the identification of single base‐pair substitutions. In addition, this method is faster and less expensive than commercial methods.
A gel retardation assay system for studying protein binding to simple repetitive DNA sequences
Authors: Mäueler Winfried , Muller Marc , Köhne Anja C. , Epplen Jörg T. 
Published: May 2005

AbstractSimple repetitive DNA sequences have been regarded as mere “junk” present in all eukaryotic genomes. In fact, mixed simple repeat (gt)n(ga)m sequences are present in major histocompatibility complex MHC‐DRB genes for long evolutionary times, including such distant animals as artiodactyla and man. We describe herein an unsophisticated method which reveals that at least certain simple repetitive (gt)n(ga)m sequences bind nuclear proteins and show characteristics of a specific DNA‐protein interaction via gel retardation.
High‐resolution capillary electrophoretic analysis of DNA in free solution
Authors: Nathakarnkitkool Suraphol , Oefner Peter J. , Bartsch Georg , Chin Mihael A. , Bonn Günther K. 
Published: May 2005

AbstractCapillary electrophoretic separations of double‐stranded DNA fragments in the size range of 20–2200 base pairs were achieved in less than 20 min with the use of a Tris‐borate buffer containing hydroxyethylcellulose. Analyses were carried out in both uncoated and phenylmethyl‐coated capillaries of fused silica with internal diameters of 50 and 100 μm, respectively, and an effective column length of 50 cm. The addition of ethidium bromide resulted in an improved resolution of double‐stranded DNA fragments, thereby permitting even the separation of fragments differing only 1–2 base pairs in length. Moreover, resolution was found to be linearly proportional to the size of the cation used to adjust ionic strength Cs+ > RB+ > K+ > Na+ > Li+. However, the analysis times also increased with increasing cation size due to a decrease in electroosmotic flow. Elution order was verified by spiking restriction digests with slab gel electrophoretically purified components. Subsequently, the described system was applied to the detection and quantitation of an mRNA transcript of the androgen receptor, which had been amplified by polymerase chain reaction and purified by size‐exclusion chromatography to avoid peak broadening due to conductivity differences between sample and running buffer. Since the actual amount of DNA introduced into the capillary cannot be defined, molar ratio–peak area ratios of the polymerase chain reaction product to various restriction fragments of known concentration were used to determine the amount of amplified DNA. The coefficient of variation was as small as 3.4% and the results were in good agreement with a spectrophotometric assay.
A single‐fluor approach to DNA sequence determination using high performance capillary electrophoresis
Authors: Pentoney Stephen L. , Konrad Kenneth D. , Kaye Wilbur 
Published: May 2005

AbstractThe Tabor and Richardson strategy for enzymatic chain termination sequencing of DNA using relative peak intensity has been adapted to high performance capillary gel electrophoresis with laser induced fluorescence detection. This approach to DNA sequencing involves the use of only a single fluor and results in significant reduction in the time required to determine a DNA sequence without the use of highly complicated and expensive instrumentation. We present a modification of the Tabor and Richardson approach employing two reactions, each containing complementary mixtures of only three ddNTP's in the concentration ratio 4:2:1. The DNA sequence is determined by relative peak height and by assigning the missing ddNTP to “gaps” between the peaks. The use of only three terminators/reaction simplifies the software task of differentiating between the termination types and makes more efficient use of the available dynamic range. Both complementary mixes generate complete sequence information and the two data files are combined in order to make a more confident sequence call. This process helps to eliminate errors caused by occasional non‐uniform incorporation of ddNTP's or false terminations and also alleviates some of the difficulty associated with reading through compressed regions of the electropherogram.
Microelectrophoresis for the separation of DNA fragments
Authors: Heller Michael J. , Tullis Richard H. 
Published: May 2005

AbstractA methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high‐resolution electronic imaging system. Separations can be carried out in either capillary tube or thin‐layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the ΦX174 DNA/ HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353–603 bp) ranged from 18 to 25 μm, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 μm to 60 μm. The ethidium bromide‐stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single‐base resolution of fluoresceinated oligonucleotides in the 20–30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double‐stranded DNA. In the extreme, single‐stranded oligonucleotides could be effectively separated in gels with pore diameters that completely excluded double‐stranded oligomers of the same length.
Ti/IrO2 as anode and Zr as cathode in multicompartment electrolyzers with Immobiline membranes
Authors: Ettori Claudia , Righetti Pier Giorgio , Comninellis Christos 
Published: May 2005

AbstractMulticompartment electrolyzers with isoelectric Immobiline membranes are used for large‐scale preparative protein purification. A series of isoelectric membranes, of defined pI values, is utilized for keeping any desired species isoelectric within each compartment of the electrolyzer. It is preferable to have electrode disks of the same surface area as the membranes for a proper performance of the instrument because electrolyte solutions of low conductivity are used. The use of Pt disks would be quite expensive; we therefore propose using Zr as a cathode and Ti/IrO2 as an anode in the electrodic compartments. This pairing of electrodes seems to give the same performance as Pt wires. Also, conventional isoelectric focusing, as well as isoelectric focusing in immobilized pH gradients, both requiring a good contact area between gel and electrodes, would benefit by using flat laminae of these metals as electrodes.
Photodestruction of fluorophores and optimum conditions for trace DNA detection by automated DNA sequencer
Authors: Kambara Hideki , Nagai Keiichi , Kawamoto Kazuko 
Published: May 2005

AbstractAlthough automated DNA sequencers are becoming popular, their sensitivity in detecting DNA bands is still around 10−17 mole/band. The sensitivity of a system depends on the laser power, labeling fluorophore, and the fluorescence‐collecting yield. The emission and photodestruction cross‐sections of the fluorophores are critical in optimizing the irradiated laser power and the migration speeds of DNA fragments to achieve high sensitivity. We investigated photodestruction cross‐sections of various fluorophores to optimize the irradiation laser power. In addition, we used a cylindrical lens system to improve the fluorescence‐collecting yield of a DNA sequencer using side entry laser irradiation. Fluoresceine isothiocyanate (FITC) commonly used in fluorescence studies, is very photo‐destructive, the cross‐section of the destruction being about 3.8 × 10−20 cm2 in buffer solution while that of Texas Red is 1.5 × 10−21 cm2. When the time for DNA fragments to transit through the irradiated region is 11 s, the optimum laser powers are 0.9 m W, with an Ar laser (488 nm) for FITC‐DNA, and 18 mW, with an He‐Ne laser (594 nm) for Texas Red DNA. We have developed a DNA sequencer, with a cylindrical lens system which improves the fluorescence‐collecting efficiency by a factor of 4, and an He‐Ne laser (5 mW). Although the sequencer uses a slab gel, an ultra‐high sensitivity of 5 × 10−20 mole/band (S/N˜4) was achieved under optimized conditions.
Fluorescence chemistries for automated primer‐directed DNA sequencing
Authors: Hawkins Trevor L. , Du Zijin , Halloran Nicolette D. , Wilson Richard K. 
Published: May 2005

AbstractA major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user‐defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primerdirected DNA sequencing, and compare and discuss the relative merits and limitations of these methods.
‘Laterally aggregated’ polyacrylamide gels for electrophoresis
Authors: Righetti Pier Giorgio , Caglio Silvia , Saracchi Marco , Quaroni Sergio 
Published: May 2005

AbstractA new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small‐ to a large‐pore gel is clearly apparent at 0.5% PEG addition and reaches a pleateau already at 2.5% PEG. Even with shorter PEG fragments (6,2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinylpyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross‐linking event) is induced via intra‐chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 μm, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site.
The effect of gel structure on matrix orientation
Authors: Stellwagen John , Stellwagen Nancy C. 
Published: May 2005

AbstractFour polymeric gels of different structure, low electroendosmosis (LE) agarose, highest electroendosmosis (HEEO) agarose, β‐carrageenan, and polyacrylamide, were studied by transient electric birefringence to determine the importance of various structural features on the orientation of the gels in an electric field. The two types of agarose, but not the polyacrylamide or β‐carrageenan, exhibited anomalous orientation effects. Both agarose and β‐carrageenan exhibited large birefringence signals, suggesting that the noncovalent hydrogen bonds joining the agarose fibers within the matrix allow the high degree of orientation of the gel. The spatial arrangement of the sugars of the agarose backbone is necessary for the anomalous orientation effects in reversing electric fields.
Agarose gel pore radii are not dependent on the casting buffer
Authors: Stellwagen Nancy C. 
Published: May 2005

AbstractPrevious studies have shown that the apparent pore size of agarose gels is dependent on the buffer in which the gel is cast and run (D.L. Holmes and N.C. Stellwagen, Electrophoresis 1990, 11, 5–15; N.C. Stellwagen and D.L. Holmes, Electrophoresis 1990, 11, 649–652). However, these studies, based on the mobility of DNA restriction fragments, neglected the effect of electroendosmosis. By measuring the mobility of vitamin B12 under various experimental conditions, it is shown here that electroendosmosis is highly buffer‐dependent. When the observed mobilities of DNA are corrected for electroendomosis, the apparent pore radii of agarose gels are found to be independent of the casting buffer.