bluuebunny/crossref_metadata_2025_split huggingface dataset:
Forensics of birds of prey by DNA fingerprinting with 32P‐labeled oligonucleotide probes
Authors: Wolfes Rüdiger , Máthé Judith , Seitz Alfred 
Published: May 2005

AbstractPaternity tests on confiscated families of eight species of birds of prey were carried out successfully by DNA fingerprinting with 32P‐labele oligonucleotide probes. Variations in the number of hybridized fragments, depend ing on the species of birds, are observed using the same probe, as well as differences of polymorphism by hybridizing the DNA samples with several oligonucleotide probes.
Monitoring genomic alterations with a panel of oligonucleotide probes specific for various simple repeat motifs
Authors: Nürnberg Peter , Barth Ingrid , Fuhrmann Eike , Lenzner Claudia , Losanova Tanka , Peters Christian , Pöche Hubert , Thiel Gundula 
Published: May 2005

AbstractGermline and somatic instability of the human genome was studied, using synthetic oligonucletides specific for simple repeat motifs. The following probes were used: (GTG)5, (GACA)4, (GATA)4, (CT)8, (TTAGGG)3, (GT)8, (GAA)6 and (GGAT)4. Each of them is unique with respect to the target regions recognized in the genome. Thus compilation of the various fingerprint data provides a complex map of the genome (and its deviations). While the fingerprints of differentiated somatic tissues never showed any alterations, in tumor tissues (namely gliomas) many changes could be detected. Most of the latter reflect secondary karyological aberrations. In nearly one third of the gliomas, drastically amplified and apparently monomorphic DNA fragments were identified. This marker should make it possible to deal with causal pathogenetic mechanisms as well as novel diagnostic strategies.
Sizing bands on autoradiograms: A study of precision for scoring DNA fingerprints
Authors: Galbraith David A. , Boag Peter T. , Gibbs H. Lisle , White Brad N. 
Published: May 2005

AbstractWe replicated DNA fingerprints of snapping turtles (Chelydra serpentina) and hypervariable restriction fragments of red‐winged black birds (Agelaius phoeniceus) to estimate the between‐blot and between‐lane components of variance in molecular weights of restriction fragments. Molecular weight standards were included in every lane, and bands were sized using a sonic digitizer. In both studies, a strong positive correlation was found between band size and coefficient of variation (CV; mean = 0.7 %). In the DNA fingerprint study, 26 % of the variance in estimates of band size was due to differences between blots, 10 % due to differences between lanes on the same blot, and 64 % due to error in the digitizing process. In the restriction fragment length polymorphism (RFLP) study, 16 % of the variance was due to difference between lanes, and 84 % to digitizing. Statistical models were developed to measure the effect of sizing error on identifying identical fragments in different lanes or on different blots, in categorizing distinct alleles, and in determining the size of bins in operational allele definitions. We suggest that the distance between bands be at least 2.8 standard deviations (SD) before they are declared different at α = 0.05, and 3.7 SD for α = 0.01. A variation in CVs strongly indicate that empirical relationships between SD and band size must be used to decide if two bands represent the same allele. Alleles must be at least 3.9 SD apart before the chance of assigning new observations in error falls below 0.05. We suggest that a minimum bin width of 16 SD is necessary before the chances of assigning a band to the wrong bin falls below 0.05.
Resolution of glycogen phosphorylase isoenzymes in precast Phastsystem polyacrylamide gels
Authors: Mayer Doris , Letsch Iris 
Published: May 2005

AbstractHomogeneous (7.5 %) and gradient (10–15 %) ultrathin nondenaturating miniaturized polyacrylamide gels (Pharmacia PhastGel media) were used to separate glycogen phosphorylase isoforms from rabbit muscle, rat liver and brain, MH 3924A cells, a dedifferentiated hepatocellular carcinoma of the rat, and C1I cells, a nontumorigenic epithelial rat liver cell line. The enzymes were detected by in situ phosphorylase assay and by immunoblotting. Phosphorylase proteins from the brain, MH 3924A, and C1I exhibited similar electrophoretic mobility, which was different from that of the enzymes from the muscle and normal liver. Molecular weight determination from sodium dodecyl sulfate gels yielded similar data for the subunits of muscle and liver enzymes (98 000 and 96 000), respectively, on one hand, and brain, MH 3924A tumor, and nontumorigenic C1I cells (93 000, 93 000 and 92 000), respectively, on the other. In the native gels a enzymes migrated as dimers: for muscle phosphorylase a, a tetramer was also observed. The a and b forms of the enzymes could not be resolved. An antibody raise against rat liver phosphorylase reacted only with the liver enzyme, whereas an antibody raised against brain phosphorylase stained the brain enzyme and the enzymes from MH 3924A and C1I cells. This indicates that hepatoma cells and immortalized nontumorigenic epithelial liver cells express a phosphorylase isoenzyme that is different from the liver type but similar to the brain type. The PhastSystem provides a rapid, sensitive, and highly reproducible method to resolve the different isoenzymes of glycogen phosphorylase.
High resolution electrophoresis of soybean seed isoesterases
Authors: Wiesner Ivo 
Published: May 2005

AbstractA modified discontinuous buffer system for vertical polyacrylamide electrophoresis was developed with large changes in component concerntration from the original Tris‐HCI/Tris‐glycine system of the Ornstein‐Davis type. The buffer system is able to resolve the fast soybean seed isoesterases crucial for cultivar discrimination. A higher number of anodic bands is obtainedin comparison with separations under standard conditions. A “gelating boundary”, resulting from the aggregation of seed proteins, is retarded and does not migrate out of the upper stacking gel. The same buffer modification is also beneficial to the separation of native seed proteins.
Two‐dimensional electrophoresis of human parotid salivary proteins from normal and connective tissue disorder subjects using immobilised pH gradients
Authors: Beeley Josie A. , Khoo Kong Soo , Lamey Philip‐John 
Published: May 2005

AbstractTwo‐dimensional electrophoretic analysis of human salivary proteins using immobilised pH gradients in the first dimension, thin‐layer gradient horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the second, and modified staining procedures has resulted in a substantial improvement in their resolution. Unlike carrier ampholyte‐based techniques, immobilised pH gradients prevent the loss of proteins of pI>8; accordingly, basic components, including basic proline‐rich proteins, can now be resolved. A two‐dimensional map showing the locations and identities of most of the major proteins has been constructed. Narrow‐range pH gradients can be constructed to give increased resolution of proteins of particular interest. By means of a pH 3.5–5.0 gradient, the abnormal salivary proteins associated with connective tissue disorders were found to be a highly heterogeneous group of pI ∼ 3.75–4.75 and Mr ∼ 32 000; although low levels occurred in some normal individuals, there was less heterogeneity (pI∼3.75–4.25). The technique should form a base for future structural, functional, and clinical studies on human salivary proteins.
Nuclear and DNA‐binding proteins in human brain tumors
Authors: Müller Andreas , Henn Wolfram , Unteregger Gerhard 
Published: May 2005

AbstractNuclear proteins obtained from human brain tumor cell lines by differential salt extraction were subjected to high‐resolution two‐dimensional electrophoresis. Several hundred spots were detectable in the low salt (0.4 M NaCl) extract using silver staining. These patterns exhibited remarkable differences between the different cell lines we analyzed. A less complex pattern occured when nuclei were subsequently treated with high salt (2.5 M NaCl/5 M urea). We compared the electropherograms from various human glioblastoma cell lines and found them very, similar and even a high degree of similarity occurs between glioblastomas and other human tumor cell lines. Beside these more general observations we detected several proteins at least enriched in human glioblastomas which were, totally absent in low grade astrocytomas and nonglial tumors. They could be separated from the bulk of nonspecific proteins by simple modifications of the isoelectric focusing conditions. From these results we conclude that nuclear proteins obtained by sequential salt extraction and separated by two‐dimensional techniques may provide tumor specific proteins suitable for antibody production.
Molecular profiles of the cell membrane bound and cytoplasmic forms of the human MHC Class‐II associated invariant polypeptides
Authors: Fernandez Nelson , Klidis Aristea 
Published: May 2005

AbstractSeveral sub‐types of histocompatibility class‐II molecules are known to exist, including DR, DQ and DP, each subset organized in alpha and beta heterodimers. These molecules play a central role in immunorecognition via direct noncovalent binding of degraded nonself immunogenic peptides followed by ligand‐interaction with T cells. In the cytoplasm the class‐II alpha and beta chains associate, in addition with a third backbone molecule known as the invariant chain. In this study we show that all class‐II subsets associate with the invariant chain probably from the same pool and that a subset of invariant chain is expressed as a cell surface subunit. This sub‐unit, whose function is still unknown, was detected by flowcytometry and by two‐dimensional gel analysis followed by antibody‐mediated immunoprecipitations.
Biochemical characterization by two‐dimensional electrophoresis of lymphocyte antigens involved in cell‐to‐cell or cell‐to‐matrix adhesion
Authors: Zocchi Maria Raffaella , Fabbri Monica , Poggi Alessandro , Gianazza Elisabetta 
Published: May 2005

AbstractWe have exemplified three cases of application of two‐dimensional (2‐D) electrophoresis to the characterization of lymphocyte membrane antigens. We could show that the proteins recognized by two monoclonal antibodies, LAK1 and LAK2, on the surface of large granular cells mediating natural‐ and lymphokineactivated killing are distinct molecules. LAK1 is expressed without any structural modification, even on the surface of endothelial cells. Another membrane antigen, recognized by the monoclonal antibody FB 12, was shown to have the overall structure of the integrins of the very late activation (VLA) class, being composed of an α and of a β subunit. The latter corresponded to the β1 type as already characterized for other VLAs, whereas the α chain was different from α1 through α6. The 2‐D protocol using immobilized pH gradients for the first dimension allows reliable assessment of the identity of individual components because of the reproducibility of the absolute coordinates for spot position.
Production of high purity proteins by elution of individual fractions from two‐dimensional gels
Authors: Tsvetkova Marina N. , Kovalyov Leonid I. , Laptev Alexei V. , Shishkin Sergei S. 
Published: May 2005

AbstractResults are presented on the production of purified myocardial proteins from two‐dimensional (2‐D) gels. Proteins were fixed in a native condition using potassium acetate and then eluted into an aqueous solution. Homogeneous tropomyosin and myosin light chain fractions and a number of nonidentified myocardial proteins present on 2‐D gels were obtained.
Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide
Authors: Holmes Diana L. , Stellwagen Nancy C. 
Published: May 2005

AbstractThe mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N′‐methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5–10.5%T (w/v); the Bis concentration ranged from 0.5–10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5–3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at ∼ 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77–90). In gels containing 3–10%C, the calculated pore radii decreased with increasing gel concentration approximately as T−0.5, suggesting that these gels may be long fiber or “1‐D” gels. The pore radii of gels with lower crosslinker concentrations decreased more slowly with increasing polyacrylamide concentration. Pore sizes were also determined for gels containing 3% Bis and 0.5% w/v agarose, and 3% Bis plus 2% w/w high molecular weight linear polyacrylamide. The pore radii of the mixed agarose‐polyacrylamide gels were only about half as large as those observed in gels of the same composition without agarose. The inclusion of 2% w/w linear polyacrylamide in the polymerization mixture decreased the effective pore size of the polyacrylamide matrix by about 10%.
Isoelectric membrane simulator: A computational approach for isoelectric Immobiline membranes
Authors: Tonani Carlo , Faupel Michel , Righetti Pier Giorgio 
Published: May 2005

AbstractIsoelectric membrane simulator (IMS) is a computer program meant for computation of pH, buffering power (β), ionic strength (I) and dissociation degree (α) of a mixture of up to 3 buffering and 1 titrant Immobilines, for generating in a reproducible manner isoelectric membranes. Such membranes, of precise isoelectric point, are then used for large‐scale protein purification by isoelectric focusing in multicompartment electrolyzers. IMS can be used, in a more general application, for titrating mixtures of buffers to a desired pH value. This versatile program is written in M. Q. BASIC rel.2.5 and it runs on any IBM hardware or compatible machine supported by MS‐DOS. An example of purification of superoxide dismutase in a multicompartment electrolyzer with a set of fixed pI membranes of widely differing composition is shown.
A modified method of genomic DNA preparation in agarose inserts for pulse field gel electrophoresis
Authors: Poddar Saibal K. 
Published: May 2005

AbstractAccording to standard protocol, DNA in agarose inserts is prepared by first embedding the Cell in agarose. This is then incubated in the required enzyme (lysozyme, lysostaphin, or zymolase) depending on the Cell type (bacterial or plants), for spheroplast formation. Subsequent treatment of the spheroplast with protein ase K allows the isolation of large genomic DNA in agarose suitable for pulse field gel Electrophoresis. An efficient and rapid method of preparation of spheroplast is described. In this method a low concentration of enzyme required for spheroplast formation was added before embedding the Cell in agarose, which facilitated the digestion of Cell wall by the enzyme and allowed use of a low amount of enzyme. Digestion of DNA in agarose inserts prepared by this method, with rare cutting restriction enzyme and pulse field gel Electrophoresis, showed that the quality of DNA was as good as obtained by the standard method.
Detection of proteins after immunoblotting on nitrocellulose using fluorescent antibodies
Authors: Abuharfeil Nizar M. , Atmeh Ragheb F. , Abo‐Shehada Mahmoud N. , El‐Sukhon Saeb N. 
Published: May 2005

AbstractA Western blot method is described utilizing 5‐dimethylaminonaphthalene‐1‐sulfonyl (dansyl chloride)‐conjugated antibodies to detect proteins transferred onto nitrocellulose. Four proteins of different molecular weight, including human albumin, gamma heavy chain, kappa and lambda light chains of immunoglobulins, were tested. Labeling with the fluorochrome was compared with the method of enzyme‐conjugated antibodies and was found to have similar sensitivity, specificity, and safety. Moreover, this method is less expensive, and the labeled antibodies can be prepared fresh in a short time and are free from the disadvantages of enzyme label whose activity is affected by the presence of activators or inhibitors in the tested specimens.
Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: Application to agarose gel and a polystyrene particle
Authors: Tietz Dietmar , Gombocz Erich , Chrambach Andreas 
Published: May 2005

AbstractThis study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle‐free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0–20% 5‐(N‐2, 3‐dihydroxypropylacetamido)2, 4, 6‐triiodo‐N,N′ bis‐(2, 3‐dihydroxypropyl)‐isophthalamide (Nycodenz). Densitometry of gelled‐in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time‐lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109–169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user‐friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135–157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle‐free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from conventional Ferguson plots. One of the possible reasons for this relatively minor discrepancy is that the particle‐free mobility changed slightly during electrophoresis, which has a different effect on electrophoresis in homogeneous gels (single time measurement) and pore gradient gels (multiple time measurements). The characterization of particles according to size and charge by pore gradient electrophoresis provides a significant operational simplification and sample economy compared to that requiring the, use of several gel concentrations, although at the price of increased requirements of instrumentation.
Implementation of a two‐dimensional electrophoresis related laboratory information processing system: Database aspects
Authors: Ali Imtiaz , Chan Yishin , Kuick Rork , Teichroew Daniel , Hanash Samir M. 
Published: May 2005

AbstractA laboratory information processing system (LIPS) has been developed to provide support for various laboratory activities related to two‐dimensional electrophoresis. In this paper we present the relational schema for each level of activity and the application development of LIPS, in terms of the relational database management system being used. We also discuss our experience with the current system.
A comprehensive two‐dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: Towards an integrated approach to the study of cell proliferation, differentiation and skin diseases
Authors: Celis Julio E. , Madsen Peder , Rasmussen Hanne H. , Leffers Henrik , Honoré Bent , Gesser Borbala , Dejgaard Kurt , Olsen Eydfinnur , Magnusson Nils , Kiil Jette , Celis Ariana , Lauridsen Jette B. , Basse Bodil , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Brandstrup Birgitte , Pedersen Peter S. , Brandt Niels J. , Puype Magda , Van Damme Josef , Vandekerckhove Joël 
Published: May 2005

AbstractA two‐dimensional (2‐D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine‐labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer‐aided 2‐D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242–254). Annotation categories reported include: “protein name” (listing 207 known proteins in alphabetical order), “basal cell markers”, “differentiation markers”, “proteins highly up‐regulated in psoriatic skin”, “microsequenced proteins” and “human autoantigens”. For reference, we have also included 2‐D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2‐D gel protein database will be updated yearly in the November issue of Electrophoresis.
Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of human parotid salivary proteins
Authors: Beeley Josie A. , Sweeney Dan , Lindsay Joan C. B. , Buchanan M. Luise , Sarna Lalita , Khoo Kong Soo 
Published: May 2005

AbstractThe proteins in human parotid saliva have been separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R‐250 and silver staining procedures have been modified to overcome the problems encountered with staining of praline‐rich proteins. By means of the CBB R‐250 procedure which stains praline‐rich proteins pink‐violet, immunoblotting, concanavalin A binding, periodate‐Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual‐to‐individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline‐rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.