bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes
Authors: Shojaee Negin , Patton Wayne F. , Chung‐Welch Nancy , Su Qin , Hechtman Herbert B. , Shepro David 
Published: May 2005

AbstractTwo principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN‐1 and FLN‐2). A protein that copurifies with an α‐naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed‐phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60—66% identity with nonmuscle filamin (ABP‐280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16‐residue synthetic peptide corresponding to a structural β‐sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non‐muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum micro‐vessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, α‐actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.
Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines
Authors: Williams Katherine , Chubb Cynthia , Huberman Eliezer , Giometti Carol S. 
Published: May 2005

AbstractHigh‐resolution two‐dimensional gel electrophoresis (2‐DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2‐DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent (56/727) of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Eight proteins present in normal cultured breast epithelial cells were not detected in any of the tumor cell lines. We identified a subset of the differentially expressed proteins using a combination of immunostaining, protein sequencing, comigration, and subcellular fractionation. These identified proteins include the intermediate filament components vimentin and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27 and hsp60 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Many of the differentially expressed proteins we identified have roles in cellular proliferation and differentiation, including annexin V, elongation initiation factor 5A, Rho GDP dissociation inhibitor, and prohibitin. We identified inosine‐5‐monophosphate dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2‐ to 20‐fold relative to the levels in normal cells. These results expand the human breast epithelial cell protein database (http://www.anl.gov/CMB/PMG) which is being built to assist researchers with the identification of abnormal patterns of expression and pathways associated with malignancy.
TMIG‐2DPAGE: A new concept of two‐dimensional gel protein database for research on aging
Authors: Toda Tosifusa , Kaji Kazuhiko , Kimura Narimichi 
Published: May 2005

AbstractCellular proteins of a normal human diploid fibroblast line (TIG‐3) at various stages of replicative aging were resolved by horizontal isoelectric focusing on an immobilized pH gradient, followed by vertical sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE). Spot proteins were visualized by silver staining and quantitated by image processing. All corresponding spots were matched among two‐dimensional gel images, and variation profiles in relative abundance of individual proteins during in vitro aging were classified into five categories, i.e., (i) increase, (ii) decrease, (iii) increase followed by decrease, (iv) decrease followed by increase, and (v) irregular or nonsignificant variation. The new concept of the Tokyo Metropolitan Institute of Gerontology two‐dimensional gel protein database (TMIG‐2DPAGE) was prepared from the above data to support research on cellular aging. The database was put on our World Wide Web home page at the URL of http://www.tmig.or.jp/2D/ to allow free access through the Internet. The individual protein data entries were linked to the standard spot protein map of the two‐dimensional gel image in order to be accessible by clicking the mouse on it.
The effects of a mixture of charged and neutral additives on analyte migration behavior in capillary electrophoresis
Authors: Kranack Andrea R. , Bowser Michael T. , Britz‐Mckibbin Philip , Chen David D. Y. 
Published: May 2005

AbstractMulticomponent additives, such as derivatized cyclodextrins with various degrees of substitution, can be considered single‐component additives as long as the fraction of each component remains constant. In this paper, equations are derived describing the effect of such additives on the migration behavior of analytes. These equations are used in the study of capillary electrophoresis (CE) systems with differentially charged cyclodextrins as additives. For weakly acidic analytes, the binding with highly negatively charged sulfobutyl ether β‐cyclodextrin (SBE‐β‐CD) increases their negative electrophoretic mobility, while the binding with neutral hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) decreases their negative mobility. By obtaining the equilibrium constants and mobilities for each additive with each analyte (in this case, phenol, 2‐naphthol and 1‐naphthol), the migration behavior of these analytes in CE systems is quantitatively predicted at various concentrations of mixtures of the two additives. The properties of the contour lines in the binding isotherm surfaces of such CE systems are discussed.
Base‐specific separation of oligodeoxynucleotides by capillary affinity gel electrophoresis
Authors: Baba Yoshinobu , Sawa Tomohiro , Kishida Akio , Akashi Mitsuru 
Published: May 2005

AbstractCapillary affinity gel electrophoresis was applied to sequence‐specific and base composition‐specific recognition of oligodeoxynucleotides, utilizing the formation of heteroduplexes between a nucleic acid analogue immobilized into the capillary gel and soluble oligodeoxynucleotides with different sequences. Capillary affinity gel electrophoresis using capillaries filled with a conjugated gel of polyacrylamide and a synthetic nucleic acid analogue [poly(9‐vinyladenine)] was effective for the selective separation of hexathymidylic acid from a mixture of four homopolymers of A6, C6, G6, and T6 and for the complete resolution of five heteropolymers of hexadeoxynucleotides (TAAAAA, TTAAAA, TTTAAA, TTTTAA, TTTTTA). We also demonstrated that capillary affinity gel electrophoresis was useful for the selective and the sensitive sequence‐specific recognition of sequence isomers of DNA (TTTTAA, TTTTAAT, TTTATA, and TTTTAA).
High‐throughput robotic system for sequencing of microbial genomes
Authors: Hilbert Helmut , Schäfer Andreas , Collasius Michael , Düsterhöft Andreas 
Published: May 2005

AbstractA high‐throughput robotic workstation system was used for double‐stranded plasmid DNA template preparation and sequencing reaction setup to streamline the sequencing process in genome projects. All 96‐well miniprep kits that were tested provided high quality plasmid DNA suitable for fluorescent DNA sequencing. After quantitation in a 96‐well UV spectrophotometer, the plasmid DNA was used as template to automatically set up sequencing reactions. The setup was controlled by spread sheets that were imported into the robotic system. We utilized this integrated system to prepare all necessary shotgun templates for our contributions to a number of large‐scale genome projects as well as a full‐length cDNA sequencing project.
Differential display approach to quantitation of environmental stimuli on bacterial gene expression (minireview)
Authors: Fislage Rainer 
Published: May 2005

AbstractThe differential display of the mRNA technique for eukaryotes is fruitful in identifying genes with altered transcription rates caused by exogenous or endogenous stimuli. Prokaryotic analogues of the method using arbitrary oligonuleotides may reach a complete statistical genome coverage. Thus a genomewide mass screening for transcriptionally regulated sequences will be possible. However, the primer sets have to be optimized for a given species to result in maximum band yields. Hence the construction of primers requires the calculation of oligonucleotide frequencey distributions from known coding regions to choose sequences with frequent occurrence in the bacterial genome. After completion of many whole genome sequencing projects, differentially regulated cDNA sequences are readily identified by sequence comparisons.
The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis
Authors: Desruisseaux Claude , Slater Gary W. , Drouin Guy 
Published: May 2005

AbstractIt has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris‐44.5 mM boric acid‐1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient.
Do DNA gel electrophoretic mobilities extrapolate to the free‐solution mobility of DNA at zero gel concentration?
Authors: Strutz Kurt , Stellwagen Nancy C. 
Published: May 2005

AbstractThe electrophoresis of small DNA fragments has been measured in dilute agarose and polyacrylamide gels cast and run in Tris‐acetate‐EDTA (TAE) and Tris‐borate‐EDTA (TBE) buffers. Ferguson plots were constructed to extrapolate the mobilities to zero gel concentration and estimate the free solution mobility of DNA. In polyacrylamide gels, in both TAE and TBE buffers, the extrapolated mobilities at zero gel concentration increased gradually with decreasing DNA molecular weight, went through a maximum at ∼ 60 bp, and then decreased again. The increase in the extrapolated mobilities with decreasing molecular weight observed for DNA fragments ⩾ 60 bp can be attributed to transient interactions between the migrating DNA molecules and the polyacrylamide gel fibers. If such interactions are eliminated by extrapolating the mobilities to both zero gel concentration and zero DNA molecular weight, the apparent free solution mobility of DNA is found to be 3.1 × 10−4 cm2V−1s−1 in TAE buffer and 4.2 × 10−4 cm2V−1s−1 in TBE buffer at 20°C, reasonably close to the actual free solution mobilities measured in the same two buffers by capillary electrophoresis (N. C. Stellwagen et al., Biopolymers 1997, 42, 687–703). The significantly larger electrophoretic mobility observed in TBE buffer is most likely due to the formation of nonspecific, highly charged deoxyriboseborate complexes in this buffer medium. For DNA molecules ⩽ 60 bp in size, the decrease in the extrapolated mobilities with decreasing molecular weight parallels the decrease in their free solution mobilities observed by capillary electrophoresis. In agarose gels, the extrapolated mobilities of small DNA molecules at zero gel concentration appear to be independent of molecular weight. The apparent free solution mobilities are found to be (3.0 ± 0.1) × 10−4 cm2V−1s−1 in TAE buffer and (3.2 ± 0.1) × 10−4 cm2V−1s−1 in TBE buffer. The very similar mobilities observed in the two buffer media suggest that the borate ions in TBE buffer primarily form complexes with the galactose residues in the agarose gel fibers, rather than with the migrating DNA molecules, because of mass action effects. The formation of borate‐agarose complexes, increasing the net negative charge of the agarose gel fibers, appears to be responsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer (N. C. Stellwagen, Electrophoresis 1992, 13, 601–603).
Enhanced hybridization labeling signals in Southern blotted DNAs fractionated with voltage gradient gel electrophoresis
Authors: Barbieri Rainer , Duro Giovanni , Izzo Vincenzo 
Published: May 2005

AbstractAn enhancement of hybridization labeling signals is demonstrated in Southern blotted DNAs, fractionated by voltage gradient gel electrophoresis. This enhancement is due to a reduced thickness of each single nucleic acid band in the gel as a consequence of the gradient effect, corresponding to an increased concentration of DNA per unit area.
Large scale isolation of genes as DNA fragment lengths by continuous elution electrophoresis through an agarose matrix
Authors: Rodriguez Henry , Akman Steven A. 
Published: May 2005

AbstractA simple procedure for the isolation of genes as DNA fragment lengths is described. By using a commercial continuous elution protein electrophoresis apparatus and incorporating an agarose matrix, preparative scale amounts (300 μg) of DNA can be purified by fragment lengths from a mixture of genomic fragment lengths with high recovery yields. Fractions corresponding to unique fragment length ranges are screened for individual genes by dot‐blot analysis. Using this technique, we have isolated two genes: PGK1, a single copy housekeeping gene; and p53 (exons 3–11), a tumor suppressor gene, whose DNA fragment lengths elute at 4 and 7.5 kbp, respectively, from a single preparative run. As an example of the utility of the technique, we applied it to improving the sensitivity of the ligation‐mediated polymerase chain reaction (LMPCR) — a nucleic acid amplification technique used for the detection and mapping of DNA damage along genes. By eliminating excess nontargeted genomic DNA, the agarose matrix continuous elution electrophoresis (CEE) procedure provided a 24‐fold increase in signal strength attributable to base damage caused by exposing DNA to reactive oxygen species. Genomic DNA fragment length purification by agarose matrix CEE should also prove useful in other research areas requiring gene isolation, such as genomics and molecular biology.
Detection of clonal T cells in cutaneous T cell lymphoma by polymerase chain reaction: Comparison of mutation detection enhancement‐polyacrylamide gel electrophoresis, temperature gradient gel electrophoresis and fragment analysis of sequencing gels
Authors: Scheller Uta , Muche J. Marcus , Sterry Wolfram , Lukowsky Ansgar 
Published: May 2005

AbstractCutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex‐loaded polyacrylamide gel electrophoresis on MDE® (mutation detection enhancement) gels (HD‐MDE PAGE), of heteroduplex‐loaded temperature gradient gel electrophoresis (HD‐TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin‐fixed, paraffin‐embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRy PCR followed by electrophoretic separation. Clonality was detected by HD‐MDE PAGE in 22, by HD‐TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD‐MDE PAGE and HD‐TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD‐TGGE and FA detected a clonal pattern down to a dilution of 103 monoclonal cells in 106 peripheral blood mononuclear cells (PBMC), while HD‐MDE PAGE revealed a detection limit of 104 monoclonal cells in 106 PBMC. In conclusion, HD‐TGGE and FA possess a higher sensitivity and lower detection limit than HD‐MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time and cost, we recommend HD‐TGGE.
Simultaneous genotyping of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci by amplified product length polymorphism (APLP) analysis
Authors: Aoshima Toshiaki , Umetsu Kazuo , Yuasa Isao , Watanabe Gotaro , Suzuki Tsuneo 
Published: May 2005

AbstractGenotyping of the alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci is important in alcohol studies. We describe a method for simulataneous genotyping of ADH2 and ALDH2 based on amplified product length polymorphism (APLP) analysis. Two polymerase chain reaction (PCR) fragments for ADH2 (57 bp, 53 bp) and two for ALDH2 (78 bp, 73 bp) are simultaneously amplified. Nine banding patterns reflecting the genotypes of the ADH2 and ALDH2 loci are clearly and unambiguously distinguished. The APLP method seems to be particularly suited for large‐scale population studies of ADH2 and ALDH2 loci because it is simple and rapid.
Population analysis of the collagen type IIα1 3′ variable number of tandem repeat polymorphism by heteroduplex genotyping
Authors: Uitterlinden André G. , Huang Qiuju , Pols Huibert A. P. , van Leeuwen Johannes P. T. M. 
Published: May 2005

AbstractThe AT‐rich variable number of tandem repeat (VNTR) marker at the 3′ end of the collagen type IIα1 (COL2A1) gene has been shown to have a complex structure with extensive sequence variations among repeat units. We analyzed this VNTR polymorphism in a large population of 972 Caucasian individuals with a genotyping procedure involving heteroduplex analysis of PCR products using polyacrylamide gel electrophoresis. Seven size alleles were identified, combining to 19 different homoduplex genotypes (heterozygosity = 0.64). These could be further dissected by analysis of heteroduplexes into 85 different heteroduplex genotypes (heterozygosity = 0.84). By systematically heteroduplexing homozygous and heterozygous individuals in vitro, characteristic heteroduplex doublet bands were generated of known allelic composition. A comparison of these doublets with heteroduplex patterns observed in the population allowed us to identify 29 alleles. The degree of correspondence with a different COL2A1 VNTR genotyping system, based on size separation of single‐strand VNTR alleles [l], was investigated by the analysis of samples typed with both methods, including samples from reference CEPH pedigrees. This revealed improved genetic resolution by the heteroduplex method including discrimination of frequent alleles considered identical by the single‐strand analysis. Our findings demonstrate heteroduplex analysis of the COL2A1 VNTR to be a robust and highly informative genetic marker system. The documented increased genetic variability has important implications in forensic and paternity testing, as well as in linkage and association studies relating genetic variation at this locus to disease endpoints.
DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary electrophoresis using replaceable linear polyacrylamide
Authors: Klepárník Karel , Berka Jan , Foret František , Doškař Jiři , Kailerová Jana , Rosypal Stanislav , Boček Petr 
Published: May 2005

AbstractThe nucleotide sequence of a part of a 4.9 kbp common restriction fragment isolated from Staphylococcus aureus bacteriophage (bacterial virus) 3A has been determined by capillary electrophoresis (CE). The fast separation of sequencing fragments in linear polyacrylamide solution at a temperature of 55°C allowed the reading of more than 650 bases of sequence in 60 min. The single strand (ss)DNA fragments were prepared by cycle sequencing with fluo‐rescently labeled dideoxy‐terminators on the cloned bacteriophage DNA template. With respect to analysis speed, sequence read‐length, low sample consumption and automation, CE offers a simple, labor‐saving and inexpensive procedure for DNA sequencing. Operating the CE columns at elevated temperature proved to be a rapid procedure capable of extending sequence read‐length. The resulting sequence of the common restriction fragment can be used for the preparation of specific primers and oligonucleotide hybridization probes for identification of Staphylococcus aureus bacteriophages and/or prophages belonging to the bacteriophage species 3A.
Enhanced separation of antidepressant drugs using a polymerized nonionic surfactant as a transient capillary coating
Authors: Harrell Crystal W. , Dey Joykrishna , Shamsi Shahab A. , Foley Joe P. , Warner Isiah M. 
Published: May 2005

AbstractThe separation of seven structurally similar antidepressant drugs (amitriptyline, nortriptyline, imipramine, desipramine, protriptyline, doxepin, and nordoxepin) was achieved in under 15 min using a novel nonionic micelle polymer, poly(n‐undecyl‐α‐D‐glucopyranoside) (PUG) by use of capillary zone electrophoresis (CZE). Systematic studies with varying polymer concentration, pH, and percent organic modifier were conducted in order to find the optimum conditions for baseline separation of the seven tricyclic antidepressants. In addition, equations for capacity factor were used to estimate the extent of what was initially thought to be micelle analyte interaction. A series of calculations show that a modified CZE system (PUG‐CZE) was the actual mode of separation. Thus, our study concluded that PUG functioned in a non‐electrokinetic chromatography mode.
High sensitivity analysis of proteins and peptides by capillary electrophoresis‐tandem mass spectrometry: Recent developments in technology and applications
Authors: Figeys Daniel , Aebersold Ruedi 
Published: May 2005

AbstractAnalytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever‐increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method. In this review article we summarize recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins. Specifically, we review recent advances in the development and application of capillary electrophoresis‐mass spectrometry (CE‐MS) and on‐line analyte concentration techniques, and introduce the emerging field of microfluidics as a front end to mass spectrometry (MS).