bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractAbnormally 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

AbstractConventional 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

AbstractThis 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

AbstractA 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

AbstractA 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

AbstractThe 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

AbstractThe 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

AbstractTen 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

AbstractThe 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

AbstractChromatographically 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

AbstractThe 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

AbstractStudies 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

AbstractThe 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

AbstractAutoimmune 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

AbstractFor 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

AbstractThis 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

AbstractDNA 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

AbstractAnalytical 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

AbstractThe 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

AbstractA 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.