bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional polyacrylamide gel electrophoresis of human lung cancer: Qualitative aspects of tissue preparation in relation to histopathology
Authors: Franzén Bo , Iwabuchi Hiroshi , Kato Harabumi , Lindholm Johan , Auer Gert
Published: May 2005

It is of great importance to identify new objective markers significant for the diagnosis, treatment, and prognosis of human cancers. Cytometric assessment of the DNA content has been shown to be one parameter which correlates excellently with the clinical course of patients with solid cancers of the breast, prostate, and thyroid. However, additional specific marker polypeptides are needed in order to further improve the diagnostic and prognostic sensitivity. Two‐dimensional polyacrylamide gel electrophoresis (2‐DE) is a unique tool in this field of cancer research, but serious problems concerning intersample variations in the 2‐DE pattern must be solved because quantitative variations of potential marker polypeptides are expected to occur at low levels. In this study we examined a modified preparation method for tumor tissues, focusing on the relation between histopathological properties and 2‐DE gel quality. We selected a group of human lung cancers sharing similar prognosis; nevertheless, the 2‐DE patterns showed significant intersample but low intrasample variations. It is concluded that histopathological features, such as a local homogeneity, and the amounts of connective tissue and serum proteins are critical factors for the successful preparation and high quality of 2‐DE‐gels.
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

AIDS 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

Skeletal 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

Poly (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

Electrophoretic 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

Polystyrene 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

An 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

The 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

An 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

Simple 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

Capillary 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

The 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

A 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

Multicompartment 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

Although 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

A 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

A 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

Four 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

Previous 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.
Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media
Authors: Wheeler David , Tietz Dietmar , Chrambach Andreas
Published: May 2005

Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to “linear” standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration (“Ferguson curves”) of such abnormally conformed DNA differ from those of “linear” standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than “linear” standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically “abnormal” DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards. Since the progressive stretching and/or orientation of DNA with increasing polymer concentration is suggested by the concave Ferguson plot of DNA and, derived from it, the plot of DNA radius vs. polymer concentration obtained for capillary zone electrophoresis data on uncrosslinked polyacrylamide and agarose solutions as well as the corresponding gels, the abnormal retardation of DNA relative to “linear” standards is being interpreted in terms of a lowered stretchability and/or orientability of abnormally conformed species as compared to “linear” ones.
PCR amplification products are of limited use for the study of DNA/protein interaction
Authors: Mäueler Winfried , Frank Gabi , Siedlaczck Ina , Epplen Jörg T. , Melmer Georg
Published: May 2005

Conventional methods for labeling double‐stranded DNA lead to high specific activity Yet they often alter the target DNA sequence to such an extent as to prevent a meaningful protein/DNA interaction analysis. Therefore we tried to establish a polymerase chain reaction (PCR)‐based method which allows radiolabeling to high specific activity and should maintain the protein binding capability of small double stranded DNA fragments. By using PCR it is possible to label double stranded DNA to high specificity, but the protein binding capability of such DNA is drastically reduced.
Pulsed‐field gel electrophoresis analysis of a Pseudomonas aeruginosa pathovar
Authors: Römling Ute , Grothues Dietmar , Koopmann Uta , Jahnke Birgit , Greipel Joachim , Tümmler Burkhard
Published: May 2005

This paper presents the genome organization and mobility of Pseudomonas aeruginosa strains that had been isolated in half‐year intervals from 30 patients with cystic fibrosis since the onset of colonization over a 2‐ to 8‐year period. The chromosomes were digested with DraI or SpeI, separated by pulsed‐field gel electrophoresis, blotted and hybridized with probes encoding housekeeping or virulence genes. Strains were differentiated by relatedness of macrorestriction fingerprints. After some turnover of strains during the first two years of colonization, each patient had acquired a set of strains that diversified during the course of the disease. In the majority of patients, two clonal lineages were found to account for colonization in the air passages but each lung habitat was characterized by some specific signature of bands in the macrorestriction fragment pattern.
Immune complexes in blood: A new strategy for the analysis of their antigen portion
Authors: Fried Roman , Wiederkehr Felix , Vonderschmitt Dieter J.
Published: May 2005

A method is described for the characterization of protein antigens from circulating immune complexes from plasma. Free immunoglobulins G were separated from larger immune complexes by gel filtration with a fast protein liquid chromatographic system. The collected immune complexes were dissociated with 4M urea into antigens and antibodies. With a second column run with 4M urea, antigens smaller than 120 kDa were separated from unloaded antibody fractions. After concentration, they were analyzed by two‐dimensional gel electrophoresis.
Towards a two‐dimensional database of common human proteins
Authors: Burggraf Dorothe , Andersson Kerstin , Eckerskorn Christoph , Lottspeich Friedrich
Published: May 2005

A protein pattern of common human proteins was constructed by comparing the two‐dimensional gel electrophoresis (2‐DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2‐DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2‐DE protein map of only common human proteins was constructed as a basis for a new 2‐DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.
Detection of the hypusine‐containing protein (HP = eIF‐5A) in crude yeast extracts by two‐dimensional Western blots
Authors: Klier Hannelore , Lottspeich Friedrich
Published: May 2005

The hypusine‐containing protein (HP) with its unique modification of a specific lysine residue resulting in the amino acid hypusine is highly conserved among all eukaryotes and is also found in Archaebacteria. Studies of the protein function in translational processes showed a stimulatory effect in the methionyl puromycin assay, but not in in vitro translation of native mRNA. It was therefore also designated as eIF‐5A. To further investigate the role of HP in cellular metabolism, we purified the protein from Saccharomyces cerevisiae and raised polyclonal antibodies in chicken. Immunoglobulin preparations from the eggs of the immunized hens were used for Western blot analysis of HP in crude yeast extracts. For those studies the soluble protein fraction of the yeast was resolved on two‐dimensional gels (first dimension: isoelectric focusing using an immobilized pH gradient (IPG), pH 4–7; second dimension: sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, 12% T) and subsequently blotted onto Fluorotrans membrane. Anodic versus cathodic application of the extracts of the IPG strips was compared.
Regulation of protein biosynthesis in neonatal rat cardiomyocytes by adrenoceptor‐stimulation: Investigations with high resolution two‐dimensional polyacrylamide gel electrophoresis
Authors: He Chang , Müller Ursula , Werdan Karl
Published: May 2005

The role of α‐ and β‐adrenoceptor‐mediated regulation of protein patterns in cultured neonatal rat cardiomyocytes was studied with high resolution two‐dimensional polyacrylamide gel electrophoresis. Spontaneously beating neonatal rat cardiomyocytes were cultured for 72 h at 37°C in serum‐free medium, supplemented with either the catecholamine norepinephrine (0.1 μM), or with norepinephrine (0.1 μM) plus the α1‐adrenoceptor blocker prazosin (1 μM), or with neither of these substances. In transmission light, 105 protein spots could be seen in the lysates from control cells, 244 spots were identified in lysates from norepinephrine‐treated cells, and 114 protein species were counted in the case of lysates from cardiomyocytes which had been cultured in the presence of norepinephrine plus prazosin. This experimental approach allows a clear classification of α‐ and non‐α (probably β)‐adrenoceptor‐mediated catecholamine effects on protein synthesis in cardiomyocytes. In comparison with previous experiments (Chang et al., Electrophoresis, this issue, pp. 748–754), less protein species were identified in the untreated control cardiomyocytes, as well as in the norepinephrine treated cells. The only actual modification in the experimental setup in these two series was the addition of dimethyl sulfoxide — a substance known for its repressive effect on oncoprotein expression — to the culture medium, as solvent for prazosin.
Genetic variability of pepper (Capsicum annuum L.) seed proteins studied by 2‐D electrophoresis with immobilized pH gradients
Authors: Posch Anton , van den Berg Bartel M. , Postel Wilhelm , Görg Angelika
Published: May 2005

Ten pepper (Capsicum annuum L.) inbred lines were successfully differentiated by two‐dimensional electrophoresis with immobilized pH gradients. Qualitative polymorphism of water‐soluble and urea/detergent‐soluble seed proteins, respectively, was investigated by computer analysis and used for establishing a dendrogram derived from maximum‐parsimony analysis. The dendrogram calculated from urea/detergent‐soluble proteins shows four types of distance indices, whereas water‐soluble proteins show two sets of inbred lines with similar intraset distance indices. The validity of the dendrograms with respect to quantitative inherited traits, such as cold tolerance and earliness, will be tested by field trials.
Stress effects on hormonal growth factors in tobacco tissues indicated by special changes in the isoelectric peroxidase patterns
Authors: Rücker Waltraud , Maier Rudolf
Published: May 2005

The isoelectric peroxidase patterns of tobacco tissue cultures allow us to draw inferences to cell elongation and cell division because certain zones in acid pH ranges respond to the influence of auxines and gibberellins (promoting cell elongation) and others respond to the influence of cytokinins (promoting cell division). Stress, due to the absence of phosphate and presence of lead in the medium causes characteristic changes in the intensity of these sensitive zones in peroxidase patterns. It may be deduced that the increase and decrease of these zones correspond to stimulation and inhibition of cell elongation and cell division, respectively. Cell elongation remains almost unaltered by lack of phosphate but is markedly inhibited by lead, while cell division is enhanced. However, stress brings about a reduction of dry weight. Reactions to stress can be observed earlier in patterns of peroxidase than in growth.
Characterization of endopolygalacturonase (EC 3.2.1.15) from Aspergillus niger as glycoprotein by electrophoretic methods and lectin affino‐blotting
Authors: Raab Barbara
Published: May 2005

Chromatographically purified endopolygalacturonase (PG) from Aspergillus niger was deglycosylated with N‐glycosidase F (PNGase F) and characterized by means of sodium dodecyl sulfate (SDS)‐electrophoresis, polyacrylamide gel electrophoresis (PAGE) without denaturing agents, isoelectric focusing (IEF) and lectin affino‐blotting. The results show that PG, which is apparently homogeneous in SDS‐PAGE but heterogeneous in IEF and PAGE, consists of at least two polypeptide chains with different glycosylation patterns. The component with the higher electrophoretic mobility is deglycosylated with PNGase F and reacts with concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA), indicating a “high mannose” or “hybrid”‐type of glycoprotein (GP). The other component may contain O‐glycosidically linked mannose, N‐acetylglucosamine or glucose.
Two‐dimensional electrophoresis of the total polypeptides in ripe red grape berries
Authors: Tesnière Catherine , Robin Jean‐Pierre
Published: May 2005

The total polypetide composition of mature grape berries was analyzed by one‐dimensional sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis and two‐dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two‐dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15000 to 110000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28000 to 83 000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28 000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.
Two‐dimensional gel electrophoresis analysis of Syrian hamster embryo cells: Morphological transformation is not cell type specific
Authors: Isfort Robert J. , Kerckaert Gary , Leigh Anderson N. , LeBœuf Robert A.
Published: May 2005

Studies were conducted to investigate the protein phenotype of normal and morphologically transformed Syrian hamster embryo (SHE) cells. Based upon two‐dimensional gel protein phenotype analysis, we conclude that (i) SHE cells are a mixture of multiple cell types including mesenchymal and epithelial cells and (ii) several cell types present in the SHE cell population can be morphologically transformed by a variety of genotoxic and non‐genotoxic carcinogens.
The gene‐protein database of Escherichia coli: Edition 5
Authors: Vanbogelen Ruth A. , Sankar Pushpam , Clark Robert L. , Bogan Jacqueline A. , Neidhardt Frederick C.
Published: May 2005

The gene‐protein database of Escherichia coli is both an index relating a gene to its protein product on two‐dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two‐dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries–a 15% increase in the number of annotated two‐dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene‐protein database is estimated to contain entries for one‐sixth of the protein‐encoding genes of E. coli.
Cell electrophoretic characterization of abnormally expanded lymphocytes in autoimmune lprcg, lpr, gld and Yaa mice, and of thymocyte subsets
Authors: Shimizu Motomu , Sekine Kiyomi , Matsuzawa Akio , Iwaguchi Takao
Published: May 2005

Autoimmune mice carrying the Iprcg/lprcg (lprcg), lpr/lpr (lpr), gld/gld (gld) and Yaa genes exhibit massive lymphoproliferation and a systemic lupus erythematosus‐like syndrome. The surface markers of abnormally expanded lymphocytes used were Thy‐l+, CD4−CD8− (double negative, DN) and CD45+ for lprcg, lpr, gld and (lprcg×gld) hybrid (F1‐lprcg‐gld) mice, and Ig+ for Yaa mice. To characterize the cell surface properties and differentiation pathway of lymphocytes in autoimmune mice, the cell electrophoretic mobility (EPM) was determined for the lymph node (LN), spleen and thymus cells. The EPM of lymphocytes derived from swollen LN was of the T cell type in lprcg, lpr, gld and F1‐lprcg‐gld mice, but of the B cell type in Yaa mice, indicating that the EPM of abnormally proliferated lymphocytes in autoimmune mice reflects their origin, and that the surface properties detected as a net negative charge were the same in abnormal and normal lymphocytes. The electrophoretic behavior of whole thymocytes was also the same in autoimmune and normal mice. The DN, and CD4+CD8− and CD4−CD8+ (single positive, SP) thymocytes from normal mice exhibited high EPM, while CD4+CD8+ (double positive, DP) thymocytes exhibited low EPM. According to the recent concept of intrathymic T cell differentiation (Schwartz, R. H., Cell. 1989, 57, 1073–1081), it is suggested that EPM of thymocytes may change with maturation in the following manner: DN thymocytes with high EPM→DP thymocytes with low EPM→SP thymocytes and autoimmune DN T cells with high EPM.
In situ alkylation of cysteine residues in a hydrophobic membrane protein immobilized on polyvinylidene difluoride membranes by electroblotting prior to microsequence and amino acid analysis
Authors: Ploug Michael , Stoffer Bjarne , Jensen Arne L.
Published: May 2005

For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (> 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and in situ alkylation after electrotransfer onto polyvinylidene difluoride membranes. Precautions against NH2‐terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.
Detection of oligoclonal IgG bands in cerebrospinal fluid by immunofixation after isoelectric focusing on polyacrylamide gels with the PhastSystem
Authors: Fredman Pam
Published: May 2005

This paper describes the detection of oligoclonal bands of IgG immunoglobulins in cerebrospinal fluids (CSF) using isoelectric focusing (IEF) and immunofixation with the PhastSystem equipment. The proteins were separated by IEF on polyacrylamide gels, pH 3–9. The IgG immunoglobulins were then fixed by overlaying the gel with antihuman IgG antiserum. Fixed immunoglobulins were detected with silver staining in the PhastSystem Development Unit. One μL of serum or CSF containing 15 mg/L of IgG was found to be optimal. The procedure had good reproducibility both with regard to IEF and silver staining of the fixed immunoglobulins. The manpower need was reduced, and the procedure has been found to be useful in the clinical laboratory.
A rapid polymerase chain reaction method for identifying fixed specimens
Authors: Gill Peter , Kimpton Cohn P. , Sullivan Kevin
Published: May 2005

DNA profiling analysis of preserved tissue samples was achieved by using an extraction method which incorporated the use of a chelating resin “Chelex” to chelate inhibitory polyvalent metal ions. An automated sequencer was used to estimate sizes of low molecular weight trimeric and tetrameric microsatellites labelled with fluorescent dyes. The entire analysis can be completed within 48h. Probabilities of chance association using two microsatellites were calculated as ranging between 10−2and 10−4. The methods described have considerable potential for use in routine genetic analysis of preserved samples.
Carbohydrate analysis of transferrin subfractions isolated by preparative isoelectric focusing in immobilized pH gradients
Authors: de Jong Gerard , van Noort Wim L. , van Eijk Hendrik G.
Published: May 2005

Analytical isoelectric focusing of human serum transferrin has revealed the presence of up to nine different transferrin subfractions. The less prevalent subfractions have hitherto not been available for analytical work on their composition. Prolonged isoelectric focusing in immobilized pH gradients is capable of effectively separating and concentrating these subfractions from other fractions that are often present in over 70‐fold excess in native preparations. Preliminary results of the carbohydrate analysis indicate a heterogeneity extending beyond the present concepts of the transferrin glycan structure.
Polyacrylamide gel electrophoresis in vertical, inverse and double‐crossing gradients of soluble polymers
Authors: Gersten Douglas M. , Bijwaard Karen E.
Published: May 2005

The presence of soluble dextrans, methylcellulose and polyethylene glycol polymers incorporated into vertical sodium dodecyl sulfate (SDS)‐polyacrylamide slabs during electrophoresis can have a pronounced effect on protein separations. The effects of various standard and inverse gradients of polymers on the electrophoretic mobility of marker proteins in 10%T, 2.66%C Laemmli‐style SDS gels, and the effects of simultaneous pore size and polymer gradients were investigated. These experiments demonstrate that the inclusion of polymers is a new, additional parameter that can be useful in resolving complex mixtures of proteins.
Concave ferguson plots of DNA fragments and convex ferguson plots of bacteriophages: Evaluation of molecular and fiber properties, using desktop computers
Authors: Tietz Dietmar , Chrambach Andreas
Published: May 2005

A desktop computer program evaluating physical properties of DNA and bacteriophages is presented. The analysis is based on data obtained from capillary and submarine‐type agarose electrophoresis. Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations. This is done conveniently by a computerized evaluation of the semi‐logarithmic plot of mobility vs. gel concentration, designated the Ferguson plot. In application to most proteins, this plot is linear and computer programs exist to evaluate it. However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA. Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose. The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer. Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.