bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional polyacrylamide gel electrophoresis in experimental hepatocarcinogenesis studies
Authors: Wirth Peter J. 
Published: May 2005

AbstractHigh resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in combination with computer‐assisted densitometry was used to analyze sequential changes in polypeptide expression during chemically (aflatoxin Bl; AFB), spontaneously, and oncogene (v‐Ha‐ras, v‐raf, and v‐raf/v‐myc)‐induced experimental rat hepatocarcinogenesis. Two‐dimensional mapping of [35S]methionine and [32P]orthophosphate‐labeled whole cell lysate and nuclear polypeptides revealed subsets of polypeptides specific for each transformation modality in the in vitro rat liver epithelial (RLE) transformation model. Many of the observed changes in whole cell lysate preparations were localized to specific subcellular organelles. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin‐ and intermediate filament‐related polypeptides (vimentin, β‐tubulin, cytokeratins 8, 14, and 18, and actin) were observed among the various transformant cell lines. Whereas alterations in the tropomyosin isoforms appeared to be transformation specific, concomitant modulation of intermediate filament expression was related more to the differentiation state of the individual cell lines than to the transformed phenotype. To integrate protein and DNA information of polypeptides believed to be critically involved during cellular transformation, N‐terminal amino acid microsequencing of selected nuclear polypeptides was performed. Preliminary results suggest that N‐terminal blockage of rat liver epithelial nuclear proteins to be minor (∼20%) with sequencing sensitivity of one pmol. These studies extend our on‐going efforts toward the establishment of computerized database of rat liver epithelial cellular proteins (Wirth et al., Electrophoresis, 1991, 12, 931–954) to aid in the delineation of polypeptides critically involved in cellular growth and differentiation as well as transformation.
The molecular chaperones HSP28, GRP78, endoplasmin, and calnexin exhibit strikingly different levels in quiescent keratinocytes as compared to their proliferating normal and transformed counterparts: cDNA cloning and expression of calnexin
Authors: Honoré Bent , Rasmussen Hanne H. , Celis Ariana , Leffers Henrik , Madsen Peder , Celis Julio E. 
Published: May 2005

AbstractWe have identified nine molecular chaperones in human keratinocytes by one or a combination of three methods: (i) reaction with antibodies raised against the purified proteins, (ii) microsequencing of two‐dimensional (2‐D) gel purified proteins, or (iii), by cloning of the cDNA and expression of its encoded protein in transformed human amnion cells using the vaccinia virus expression system. The expression levels of each of the molecular chaperones were analyzed in quiescent, normal proliferating, and simian virus SV40 transformed K14 keratinocytes by cutting the corresponding protein spots from dried 2‐D gels and counting the radioactivity by liquid scintillation. The most striking observation was the strong up‐regulation (936%) of the small heat shock protein HSP28 in the quiescent keratinocytes, a fact that is in line with recent data indicating that the murine homologue (HSP25) may act as a growth inhibitor. Several chaperones that localize to the endoplasmic reticulum and that are involved in the secretory pathway (GRP78, GRP78v, endoplasmin, and calnexin) were expressed at approximately similar levels in normal proliferating and K14 keratinocytes but were down‐regulated by 50% or more in the quiescent cells, implying that these cells may possess an impaired ability to secrete certain proteins. Both GRP78 and endoplasmin genes have similar sequences in the promoter regions, suggesting that they may be partly co‐regulated at the transcriptional level (McCauliffe et al., J. Biol. Chem. 1992, 267, 2557–2562). Whether this also applies to the calnexin promoter region must await cloning of the gene which is now possible with the human cDNA reported here. Only three chaperones were up‐regulated in K14 (HSP60, HSC70, and HSP90), and their increased expression may be related to the transformed phenotype.
Complex protein composition of isolated focal adhesions: A two‐dimensional gel and database analysis
Authors: Niederreiter Markus , Gimona Mario , Streichsbier Franz , Celis Julio E. , Small J. Victor 
Published: May 2005

AbstractCurrent ideas about the composition of the focal adhesion complexes in cultured cells are based mainly on indirect immunocytochemical data. We here report a two‐dimensional (2‐D) gel electrophoresis analysis of the focal adhesion associated‐structures that remain in the growth substrate after removal of cells by mechanical shearing. Many proteins additional to the known adhesion proteins, and in higher abundance, could be identified. Using selective extraction procedures, employing detergent or gelsolin, these could be classified as either membrane‐associated, actin‐associated or both. Cross correlation of these polypeptode patterns with a 2‐D gel database allowed identification of some proteins, not previously considered as resident of focal adhesions. The data point to a more complex make up of focal adhesions than formerly supposed.
Reference points for comparisons of two‐dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions
Authors: Bjellqvist Bengt , Basse Bodil , Olsen Eydfinnur , Celis Julio E. 
Published: May 2005

AbstractA highly reproducible, commercial and nonlinear, wide‐range immobilized pH gradient (IPG) was used to generate two‐dimensional (2‐D) gel maps of [35S]methionine‐labeled proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2‐D gel protein database were identified in the 2‐D gel maps and their isoelectric points (pI) were determined using narrow‐range IPGs. The latter established a pH scale that allowed comparisons between 2‐D gel maps generated either with other IPGs in the first dimension or with different human protein samples. Of the 41 proteins identified, a subset of 18 was defined as suitable to evaluate the correlation between calculated and experimental pI values for polypeptides with known composition. The variance calculated for the discrepancies between calculated and experimental pI values for these proteins was 0.001 pH units. Comparison of the values by the t‐test for dependent samples (paired test) gave a p‐level of 0.49, indicating that there is no significant difference between the calculated and experimental pI values. The precision of the calculated values depended on the buffer capacity of the proteins, and on average, it improved with increased buffer capacity. As shown here, the widely available information on protein sequences cannot, a priori, be assumed to be sufficient for calculating pI values because post‐translational modifications, in particular N‐terminal blockage, pose a major problem. Of the 36 proteins analyzed in this study, 18–20 were found to be N‐terminally blocked and of these only 6 were indicated as such in databases. The probability of N‐terminal blockage depended on the nature of the N‐terminal group. Twenty six of the preteins had either M, S or A as N‐terminal amino acids and of these 17–19 were blocked. Only 1 in 10 proteins containing other N‐terminal groups were blocked.
Distinction of weakly homologous cDNA amplificates by single‐strand conformation polymorphism analysis: Application to guanylyl cyclase isozymes
Authors: Krause Günter , David Sigrid , Büchler Wolfgang , Wehling Martin , Gerzer Rupert 
Published: May 2005

AbstractBy use of the polymerase chain reaction (PCR), uniform amplification products of 225 to 240 bp length were obtained from five cDNA clones representing different types of guanylyl cyclases. These short DNA double strands were differentiated by single‐strand conformation polymorphism (SSCP), using polyacrylamide gel electrophoresis with the Pharmacia Phast‐System. Following heat denaturation, the samples were separated on native polyacrylamide gels at different running temperatures. Nucleic acids on the gel were detected by an automated silver stain procedure. Using 7.5% homogeneous or 4–15% gradient polyacrylamide gels at a temperature of 12°C, single‐strand conformations of amplificates, representing three different particulate guanylyl cyclases and the two subunits of soluble guanylyl cyclase, were differentiated. The characteristic banding patterns resulting from dissimilar migration of the single‐strand conformations were assigned to different guanylyl cyclase types. For the enzyme family of guanylyl cyclases, the feasibility of a combined PCR and electrophoresis approach for analyzing the expression of related genes was demonstrated. This application of the PCR‐SSCP technique provided a rapid and sensitive tool for the characterization of PCR products obtained with a common primer pair and suggested its use for investigating the tissue distribution of gene expression within a class of homologous proteins.
Classification of wheat varieties by isoelectric focusing patterns of gliadins and neural network
Authors: Søndergaard Ib , Jensen Kirsten , Krath Britta Naimi 
Published: May 2005

AbstractClassification of wheat varieties, using isoelectric focusing patterns of the gliadins, image processing and neural networks, is described. The method was compared to a statistical classification method, discriminant analysis. The isoelectric point and the area of each band were calculated by image processing. Different methods of presenting the electrophoretic patterns to the neural network were studied. The most effective method was transformation of the electrophoretic pattern to a small (11 × 47 pixels) representation of the original digitized image, which was presented to the neural network as a vector. The neural network was trained with a number of patterns and tested with new patterns from different electrophoretic runs of the same wheat varieties. In this study we used ten different wheat varieties and the neural network was able to classify 95.5% of the patterns correctly. The statistical classification method classified the same data set 91.8% correctly. We conclude that both the neural network and discriminant analysis were able to classify the patterns correctly with a high degree of certainty. The patterns that were misclassified were indistinguishable by visual inspection.
Rapid method for isolating detergent‐insoluble outer membrane proteins from Pseudomonas aeruginosa
Authors: Ravaoarinoro Madeleine , Ciurli Cristina , Toma Emil , Morisset Richard 
Published: May 2005

AbstractThe present study compares two techniques for isolating outer membrane proteins (OMPs) of Pseudomonas aeruginosa, Method A ‐ selective solubilization with sodium lauryl sarcosinate, and Method B ‐ isopycnic sucrose gradient centrifugation, using three criteria: the amount of proteins obtained, polypeptide patterns after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and their practical aspects. Method A appears to be superior to Method B. It yields a higher outer membrane protein content and a similar polypeptide pattern as Method B, but is more rapid and less cumbersome.
A transient entanglement coupling mechanism for DNA separation by capillary electrophoresis in ultradilute polymer solutions
Authors: Barron Annelise E. , Blanch Harvey W. , Soane David S. 
Published: May 2005

AbstractUsing capillary electrophoresis, large DNA molecules (2.0–23.1 kbp) may be rapidly separated in ultradilute polymer solutions (< 0.002% w/w) under, a high‐voltage, steady field (265 V/cm). At this polymer concentration, the separation mechanism appears to be significantly different from that postulated to occur in crosslinked gels. Based on experimental results obtained with DNA restriction fragments and with negatively charged latex microspheres, we conclude that the Ogston and reptation models typically used to describe gel electrophoresis are not appropriate for DNA separations in such dilute polymer solutions. Electrophoresis experiments employing solutions of both small and large hydroxyethyl cellulose polymers highlight the importance of polymer length and concentration for the optimum resolution of DNA fragments varying in size from 72 bp to 23.1 kbp. A transient entanglement coupling mechanism for DNA separation in dilute polymer solutions is developed, which suggests that there is no a priori upper size limit to DNA that can be separated by capillary electrophoresis in a constant field.
Capillary zone electrophoresis of derivatized acidic monosaccharides
Authors: Mechref Yehia , El Rassi Ziad 
Published: May 2005

AbstractA new and specific precolumn derivatization reaction for acidic monosaccharides was introduced and evaluated in the separation and sensitive detection of carbohydrates by capillary electrophoresis. The derivatization reaction involved the attachment of sulfanilic acid (a UV absorbing tag) or 7‐aminonaphthalene‐1,3‐disulfonic acid (a UV absorbing and fluorescing tag) via a condensation reaction between the amino group of the derivatizing agent and the carboxyl group of the sugar in the presence of a water‐soluble carbodiimide. The derivatization reaction replaced the weak carboxylic acid of the sugar by a strong sulfonic acid, which is fully ionized at all pH. This allowed the electrophoresis of the sugar derivatives over a wide pH range and permitted the determination of acidic carbohydrates at very low femtomole levels by UV and fluorescence detection.
A new method for the detection of proteolytic activity in Pseudomonas lundensis after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Lundy Fionnuala T. , Magee Anthony C. , Blair Ian S. , McDowell David A. 
Published: May 2005

AbstractA new method for the visualization of proteolytic activity in cell culture supernatant from Pseudomonas lundensis after sodium dodecyl sulfate (SDS) – gel electrophoresis is described. Following conventional electrophoresis, the gel is washed in a methanol‐containing buffer to facilitate partial removal of SDS. After incubation with 0.5% casein the gel is stained for protein with Coomassie Brilliant Blue R‐250. Bands with proteolytic activity appear as clear areas in the gel against a blue‐stained background. Molecular weight standards electrophoresed in the same gel stain more intensely than the background and allow determination of the molecular weights of the proteolytic components. The sensitivity of post‐electrophoretic reactivation in SDS‐gels was determined using trypsin as standard. A slight modification of the technique allowed detection of proteolytic activity in nondenaturing and in isoelectric focusing gels.
Surface‐charge reversed capillary zone electrophoresis of inorganic and organic anions
Authors: Oefner Peter J. 
Published: May 2005

AbstractAnions commonly found in biological fluids such as chloride, citrate, phosphate, bicarbonate, lactate, aspartate and glutamate, were determined within 60 s by means of capillary zone electrophoresis and indirect UV detection using 10 mM of chromate, pH 8.0, as both carrier electrolyte and chromophore. The lower mass detection limit was 0.5 pmol. In order to avoid a gradual shift of the pH and, in consequence, drifting ionic mobilities due to the formation of hydroxyl ions at the cathode, 1 mM of 5,5‐diethylbarbiturate was added to the background electrolyte. Moreover, fused silica capillaries were coated with a 0.0003% solution of hexadimethrine bromide for two minutes between runs to reverse electroendosmotic flow which otherwise would counteract the electrophoretic migration of the anions and, hence, increase time of analysis. It is concluded that charge‐reversed capillary zone electrophoresis in concert with indirect UV detection is superior to ion chromatography and conductivity detection in the determination of inorganic and organic anions in biological fluids both with regard to selectivity, sensitivity and speed of analysisPresented in part at the International Ion Chromatography Symposium, Linz, Austria, September 21–24, 1992
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Detection of platinum species in plant material
Authors: Messerschmidt Jürgen , Alt Friedrich , Tölg Günther 
Published: May 2005

AbstractModel experiments for the detection of platinum species in extracts from native and platinum‐treated grass cultivations are described. The procedural steps are cultivation of the grass samples, extraction and concentration of the platinum species by ultrafiltration and freeze‐drying, preparative separation of the species by gel chromatography followed by isotachophoresis, and sequential analytical detection of the separated platinum species by adsorptive voltammetry. After isotachophoresis, sharp peaks of platinum species could be detected. In the native grass extract only one platinum species (160–200 kDa) was found. In the platinum‐treated grass extracts several platinum species were observed in the molecular mass range from 1 to > 1000 kDa. By an extremely sensitive platinum determination method (adsorptive voltammetry; detection limit, 2 pg Pt abs.) it was possible to detect platinum even in stained protein bands from horizontal gel electrophoresis of platinum containing fractions obtained after isotachophoresis.
Detection of immunoglobulin G glycosylation changes in patients with rheumatoid arthritis by means of isoelectric focusing and lectin‐affinoblotting
Authors: Hänsler Marion , Kötz Kerstin , Häntzschel Holm 
Published: May 2005

AbstractPatients with rheumatoid arthritis have a reduced prevalence of immunoglobulin G (IgG) oligosaccharide chains terminating in galactose, thus exposing N‐acetylglucosamine. We analyzed IgG glycosylation in patients with rheumatoid arthritis, patients with early synovitis, and in controls by means of isoelectric focusing and lectin‐affinoblotting. The ratio of N‐terminal N‐acetylglucosamine and galactose was determined using specific biotin‐labeled lectins. The IgG glycosylation state may well be of clinical value in the differential diagnosis of patients presenting with early synovitis.
Mineral‐bound noncollagenous proteins in archaeological human skeletons
Authors: Freundorfer Stephan , Grupe Gisela , Weickmann Dirk 
Published: May 2005

AbstractArchaeometric approaches to archaelogical human bone also include the extraction, identification and molecular analysis of surviving bone proteins. Due to its abundance as a matrix protein, most studies focus on collagen (e.g. radiocarbon dating). Also, a variety of serum proteins are detectable in excavated skeletons. Very limited knowledge still exists on mineral‐bound noncollagenous bone proteins from ancient bones because, in the mature tissue, they occur in trace amounts only. Moreover, post‐mortem decomposition is likely to change characteristic features of the molecules. Due to their suggested role as growth and developmental factors, identification and quantification of such proteins should be valuable for both physical anthropology and epidemiology. We present a valid method for the detection of small amounts of surviving mineral‐bound noncollagenous proteins in excavated human bones up to 7500 years of age.
Theory of the mobility‐shift assay of nonspecific protein‐DNA complexes governed by conditional probabilities: The HU:DNA complex
Authors: Cann John R. , Pfenninger Oswald , Pettijohn David E. 
Published: May 2005

AbstractComplexes of an 88 bp DNA and the HU protein were studied by both experimental and theoretical electrophoretic mobility‐shift analyses. Experimental analysis defined the stoichiometry of binding and estimated an apparent intrinsic dissociation constant (Kd = 1 to 3 × 10−7 M) for the HU:DNA complexes. The theory of conditional probabilities was applied to the binding of HU to DNA in order to fix the initial equilibrium composition of mixtures to be assayed theoretically by the mobility‐shift procedure. Electrophoretic mobility‐shift patterns were obtained by numerical solution of a set of simultaneous transport‐reaction equations, in which the chemical kinetic term is formulated in terms of dissociation of the different DNA:HU complexes in gel cages. The computed patterns simulated the experimental patterns describing the titration of a fixed concentration of an 88 bp DNA fragment with dimeric HU. These insightful results provide guidelines for interpretation of the electrophoretic behavior of systems in which a ligand binds nonspecifically to DNA. In particular, the narrow unresolved zone observed both experimentally and theoretically beyond 50–60% saturation is a reaction zone characteristic of noncooperative ligand‐binding governed by conditional probabilities. The discrepancy between the theoretically assigned and experimental values of the intrinsic binding constant is attributed to an HU‐induced change in the conformation of DNA.
A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate‐polyacrylamide gels
Authors: Swain Marianne , Ross Neil W. 
Published: May 2005

AbstractA simple, sensitive silver staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis has been developed which yields high resolution of proteins with a transparent background. The method includes the incorporation of a water wash after a short fixation step in ethanol/acetic acid and prior to glutaraldehyde cross‐linking, which appears to be necessary for the high resolution staining of protein bands and the low background staining.
Dynamics of peak dispersion in capillary zone electrophoresis including wall adsorption I. Theoretical model and results of simulation
Authors: Gaš Bohuslav , Štědrý Milan , Rizzi Andreas , Kenndler Ernst 
Published: May 2005

AbstractA mathematical model is described for the simulation of peak profiles in capillary zone electrophoresis taking wall adsorption into account. It is based on such physico‐chemical relations as mass balance equations, sorption rate equations and appropriate boundary conditions. The numerical solution of the model is carried out, allowing the depiction of the concentration profile of the sample in the capillary as a result of the dynamics of the processes involved, which depend on a number of input parameters: rate constants, adsorption isotherms, column dimensions, field strength etc. Four cases are discussed in detail, namely those with either slow or fast adsorption kinetics and linear or nonlinear isotherms.
Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary electrophoresis
Authors: Butler John M. , McCord Bruce R. , Jung Janet M. , Lee James A. , Budowle Bruce , Allen Ralph O. 
Published: May 2005

AbstractCapillary electrophoresis (CE) is an analytical technique which provides rapid, high resolution analysis of amplified DNA fragments produced by the polymerase chain reaction (PCR). In this study, two internal standards are used as size markers to bracket und precisely size PCR products. The technique is applied to typing PCR products from the short tandem repeat locus HUMTH01. HUMTH01 consists of five to seven major alleles in the size range of 179–203 bp, with each allele four bp apart. Using this genetic marker, a population containing 97 individuals was examined with both polyacrylamide gel electrophoresis and CE. Identical genotypes were obtained with both techniques demonstrating the reliability of CE in DNA typing applications. The DNA analysis took place in sets of 10 with a calibration of the CE being performed between each set of samples. For the 97 samples examined, the pooled standard deviation was 0.3 bp. The observed genotype frequencies determined from the sample set did not deviate significantly from Hardy‐Weinberg expectations. From these CE results, we conclude that HUMTH01 PCR products can be accurately and precisely sized by capillary electrophoresis using the method described.