bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional electrophoretic studies on down‐regulated intracellular transferrin in human fibroblasts immortalized by treatment with either 4‐nitroquinoline 1‐oxide or 60Co gamma rays
Authors: Kondo Tadashi , Mihara Koichiro , Inoue Yusuke , Namba Masayosi 
Published: May 2005

AbstractThe immortalization of human cells is an important early step of carcinogenesis. To investigate the mechanisms of immortalization, we established three immortalized human fibroblast cell lines by treatment with either 4‐nitroquinoline 1‐oxide (4NQO) or 60Co gama rays. Using two‐dimensional gel electrophoresis (2‐D PAGE), we identified proteins which are down‐regulated in these immortalized cell lines. Their isoelectric points ranged between 5.5 and 6.3, and their molecular masses were approximately 80 kDa. The proteins were also decreased in another human fibroblast cell line immortalized with simian virus 40 (SV40) and two human cervical cell lines. The proteins were present in a cytoskeletal fraction, reacted with anti‐transferrin antibody and some of them were phosphorylated. The proteins were identified as transferrin. Although the 2‐D PAGE patterns of our proteins and serum transferrin of different origin were distinct from each other, their isoelectric points and molecular weights were similar. We implicate some functions of intracellular transferrin, other than an iron transporter, in immortalization of human cells and discuss a new aspect of transferrin metabolism in the cells.
Multiple capillary DNA sequencer that uses fiber‐optic illumination and detection
Authors: Quesada Mark A. , Zhang Shiping 
Published: May 2005

AbstractAn 8‐capillary prototype electrophoresis system for DNA sequencing has been construted. The sequence of 400–450 bases can be obtained from each capillary in less than an hour form sequencing reactions generated with four‐color fluorescent terminators. Illumination of each capillary and collection of fluorescence is through individual optical fibers. Resolution of the DNA ladder is through a replaceable sieving matrix of linear polyacrylamide in reusable coated capillaries. Light from an argon ion laser is introduced into a fused biconically tapered fiber‐optic splitter, and individual fibers deliver approximately 10 mW of 514 nm light to each of the eight electrophoresis capillaries. Illumination and collection are by fibers normal to the surface of the electrophoresis capillary and at right angle to each other. Illumination by a fiber with low numerical aperture and collection by a fiber with high numerical aperture provides good sensitivity and signal‐to‐noise ratios without the need for microlenses (limit of detection: 1.5 × 10−11 M fluorescein analog dye with a signal‐to‐noise ratio of 2). The eight collection fibers are passed in parallel through holographic filters for Rayleigh rejection and into an imaging spectrograph, which simultaneously displays the full fluorsecence spectrum (475–648 nm) from the eight capillaries in parallel on the surface of an intensified chargecoupled device (CCD). The CCD is read out at a rate of 3.4 complete images per second.
The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices
Authors: Klepárnik Karel , Foret František , Berka Jan , Goetzinger Wolfgang , Miller Arthur W. , Karger Barry L. 
Published: May 2005

AbstractCapillary electrophoresis with a replaceable linear polyacrylamide matrix operated at elevated column temperatures of 55° and 60°C was used to exted the separation of DNA sequencing fragments to lengths greater than 800 bases. A solid‐state heater was employed to provide stable, uniform temperature control over a significant portion of the capillary. The polymer matrix, 3% w/v linear polyacrylamide in a denaturing buffer, was replaced in the capillary after each run. Using dye‐labeled primers and Sequenase chemistry on an M13mp18 single‐stranded template, four‐color separations for the sequencing products were obtained, with read lengths in excess of 800 bases. This paper also briefly discusses the effects of buffer denaturants and capillary temperature on separation speed, resolution, and gel compression.
Fast capillary electrophoresis‐laser induced fluorescence analysis of ligase chain reaction products: Human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy
Authors: Muth Jochen , Williams P. Mickey , Williams Stephen J. , Brown Michael D. , Wallace Douglas C. , Karger Barry L. 
Published: May 2005

AbstractHigh speed capillary electrophoresis‐laser‐induced fluorescence (CE‐LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA‐ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Blusescript® II phagemid. LCR‐CE‐LIF was then applied to detect point mutations in human mitochondrial DNA. resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatale.
Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis
Authors: Cossu Gianfranco , Angius Antonia , Gelfi Cecilia , Righetti Pier Giorgio 
Published: May 2005

AbstractA safe and reliable method for determining RhD type (positive or negative) and zygosity (D/D or D/d) could have applications, for instance, in the prediction of the D genotype of a father in couples where there is an RhD‐negative woman at risk of fetal alloimmunization. Capillary zone electrophoresis (CZE) is proposed as a novel, reliable and powerful method for quantitative evaluation of polymerase chain reaction (PCR) products. RhD is determined by amplifying a 136 bp region common to the RhCcEe and RhD genes and a 186 bp region specific of the RhD gene. RhD positive and negative samples are identified by polyacrylamide gel slab electrophoresis, followed by silver staining of the DNA bands, and by CZE in sieving liquid polymers, with direct on‐line peak densitometric evaluation by exploiting the intrinsic UV absorbance of the DNA fragments at 254 nm. The CZE method allows not only a reliable assessment of the RhD type (the presence of both 136 bp and 186 bp fragments indicating an RhD‐positive type, the presence of only the 136 bp fragment indicating an RhD‐negative type), but also a rapid determination of the zygosity based on the quantitative expression ratio of the 136 bp/186 bp pair. Thus, a 2:1 peak ratio clearly indicates a D/D homozygous individual, whereas a 3:1 peak ratio gives evidence of a D/d heterozygous individual.
Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis
Authors: Kilár Ferenc 
Published: May 2005

AbstractStereoselective interaction of drugs with human serum transferrin in capillary zone electrophoresis is described. The substances passed a pseudo‐stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Drugs with different structures were screened and the enantiomers of bupivacaine, propranolol and promethazine as well as the diastereomers of labetalol were resolved. Racemic mixtures of atenolol and pindolol enantiomers could not be resolved under these conditions.
Application of capillary zone electrophoresis for analysis of thyreostatics
Authors: Křivánková Ludmilla , Krásenský Simon , Boček Petr 
Published: May 2005

AbstractCapillary zone electrophoresis was optimized for the separation of thiouracil, methylthiouracil and propylthiouracil. Methylthiouracil could be determined in various types of urine (human, bovine, horse), either without any pretreatment or in ethyl acetate extracts, within 15 min. For identification, the simultaneous detection at three UV wavelengths (216, 245 and 278 nm) was advantageously used while for quantification the wavelength of the absorbance maximum at 278 nm was preferred. Under optimized conditions a linear response of the detector in the concentration range 0.1–100 ppm was obtained. On analysis of untreated urine, a detection limit of 0.5 ppm was found; for urine extracts the detection limit was 0.1 ppm. Univocal peak identification, based on absorption at three wavelength, was only possible above 2 ppm. Relative standard deviation for standard solutions of methylthiouracil, diluted in the background electrolyte, was 1%; for methylthiouracil in extracts dissolved in the background electrolyte it was 4.5%, and for methylthiouracil in untreated urine, 12.7%.
Data‐adaptive algorithms for calling alleles in repeat polymorphisms
Authors: Stoughton Roland , Bumgarner Roger , Frederick William J. , McIndoe Richard A. 
Published: May 2005

AbstractData‐adaptive algorithms are presented for separating overlapping signatures of heterozygotic allele pairs in electrophoresis data. Application is demonstrated for human microsatellite CA‐repeat polymorphisms in LiCor 4000 and ABI 373 data. The algorithms allow overlapping alleles to be called correctly in almost every case where a trained observer could do so, and provide a fast automated objective alternative to human reading of the gels. The algorithm also supplies an indication of confidence level which can be used to flag marginal cases for verification by eye, or as input to later stages of statistical analysis.
Screening for renal carcinoma associated mutations in the von Hippel‐Lindau tumor suppressor gene by temperature gradient gel electrophoresis
Authors: Wenzel Michael , Enczmann Jürgen , Uhrberg Markus , Hernández Ana , Wiese Ulrich , Ackermann Rolf , Schmitz‐Draeger Bernd , Ebert Thomas , Wernet Peter 
Published: May 2005

AbstractRenal cell carcinoma is the most common neoplastic disease of the adult kidney and occurs in its sporadic and hereditary form. Approximately 57% of all renal carcinomas of the clear cell type analyzed revealed a mutation in the von Hippel‐Lindau disease (VHL) gene. In the present work, temperature gradient gel electrophoresis (TGGE) is presented as a rapid and precise polymerase chain reaction (PCR)‐employing methodology for the detection of mutations in the VHL gene. The theoretical efficiency of TGGE to detect mutations in every base pair of the gene was calculated. According to computer analysis, at least 92% of all known mutations in the VHL gene are detectable. This calculated figure appears to be in agreement with the experimental results. Primary difficulties in analyzing exon 1 of the VHL gene were overcome by the employment of psoralen‐cross‐linked PCR fragments. In addition, TGGE analysis was used in screening for possible mutations in thirteen renal carcinoma samples. With this protocol TGGE is successfully added to the array of methods for the screening of VHL mutations.
Translationally controlled tumor protein: A protein identified in several nontumoral cells including erythrocytes
Authors: Sanchez Jean‐Charles , Schaller Dominique , Ravier Florence , Golaz Olivier , Jaccoud Sylviane , Belet Monique , Wilkins Marc R. , James Richard , Deshusses Jacques , Hochstrasser Denis 
Published: May 2005

AbstractThe translationally controlled tumor protein (TCTP) is a growth‐related protein which is regulated at the translational level. It is present in mammals, higher plants and Saccharomyces cerevisiae. This study was undertaken to localize and further characterize the TCTP in human cell lysates using two‐dimensional gel electrophoresis, monoclonal antibodies, and 45Ca‐gel overlay. TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas. It could not be detected in kidney and renal cell carcinoma (RCC). A monoclonal antibody raised against TCTP detected three isoforms likely due to post‐translational modifications. A calcium binding property was found as well as heat stability and cytoplasmic localization. The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function.
Capillary electrophoresis of organotin compounds with indirect UV detection
Authors: Whang Kuo‐Shu , Whang Chen‐Wen 
Published: May 2005

AbstractA capillary electrophoresis (CE)‐indirect UV absorbance detection method for organotin compounds is described. The five triorganotin cations, viz., trimethyltin (TMT), triethyltin (TET), tripropyltin (TPT), tributyltin (TBT) and triphenyltin (TPhT), were efficiently separated by CE in 10 min with a 5 mM acetate buffer (pH 4.5) containing 3 mM 4‐aminopyridine (4‐AP). 4‐AP also functioned as a UV‐absorbing coion for the indirect detection of these UV‐transparent organotin species. Simultaneous separation of di‐ and triorganotins was achieved with the addition of α‐cyclodextrin (α‐CD) as a modifier in the electrophoresis buffer. Linearity (r≥ 0.995) of about two orders of magnitude was generally obtained. The concentration limits of detection for the organotin compounds studied were between 2 and 20 μM (as tin). The relative standard deviation (RSD) values were in the range 1.3–7.1%. The applicability of the method for the analysis of marine sediment was examined.
Identification of mouse liver proteins on two‐dimensional electrophoresis gels by matrix‐assisted laser desorption/ionization mass spectrometry ofin situenzymatic digests
Authors: O'Connell Kathy L. , Stults John T. 
Published: May 2005

AbstractA number of proteins from a silver‐stained two‐dimensional (2‐D) electrophoresis gel of mouse liver whole‐cell lysate were identified by peptide mass mapping and sequene database searching. The excised protein spots were processed byin situreduction and alkylation, followed by Lys‐C digestion. The masses of the resulting peptide mixtures were measured with a matrix‐assisted laser desorption/ionization (MALDI) reflectron‐time‐of‐flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post‐source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.
Convergence of amino acid compositions of certain groups of proteins aids in their identification on two‐dimensional electrophoresis gels
Authors: Galat Andrzej , Rioux Vincent 
Published: May 2005

AbstractThe amino acid composition (AAC) versus the protein identity (PI) method was used for establishment of the identities of proteins from bovine brain and kidneys which were prefractionated on a CM52 cation exchanger and by preparative flat‐bed isoelectric focusing. Established identities of proteins whose AACs converge with those of other members of their proper superfamily are reliable. Groups of convergent AACs can be extracted from protein databases using the standard root‐mean‐square rule (Rmsd) with measures the difference between the AAC of chosen protein versus those in the database. Convergence of AACs of proteins is dependent on several factors such as the upper limit of Rmsd, the limits of variations of molecular mass (m) and isoelectric point (pI), the number of proteins with similar AACs present in protein databases, and the domain structure of proteins. AACs of many proteins remain unique if the Rmsd is maintained with 1.5–1.0 with m ± 3kDA and pI ± 4. Certain groups of multidomain proteins have quasi‐unique AACs only if the Rmsd is restrained to a value within 1.0 and 0.7. Convergence of AACs of certain groups of proteins may indicate that a common biological function exists for some members of each group. The AAC‐PI method may become an additional search tool for protein functions.
An on‐line two‐dimensional polyacrylamide gel electrophoresis protein database of adult Drosophila melanogaster
Authors: Ericsson Christer , Pethö Zoltàn , Mehlin Hans 
Published: May 2005

AbstractAn annotated two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) protein database of adult Drosophila melanogaster has been constructed, based on the protein patterns of heads, thoraces and abdomens of adult male and female Drosophila melanogaster. About 1200 major protein spots are catalogued. Common proteins, found in all body parts, as well as bodypart‐ and sex‐specifically expressed proteins are reported. Of the major proteins, 91, or 7.5%, are differentially expressed in the two sexes or in different body parts, at least in part reflecting specific functional requirements. At the present time 43 proteins, or about 3.5% of the detected proteins, have been identified. These data can be accessed interactively from our World Wide Web (WWW) server through clickable inline gel images and hypertext links. Identified protein spots are cross‐referenced, through hypertext links, to the SWISS‐PROT annotated database of protein primary sequences and the Fly‐Base database of Drosophila genomic data. Our reference gels can be used to gain immediate access to protein spot identify and to the pattern of differentially expressed proteins in Drosophila melanogaster. The work presented in this article ties together information from protein 2‐D PAGE, molecular biology and geneties and offers a uniform way to access this large volume of data.
“Active” two‐dimensional electrophoresis of rat liver DNA‐polymerase α
Authors: Naryzhny Stanislav N. 
Published: May 2005

AbstractThe approach of so‐called active gel analysis was used to determine the position and appearance of the catalytic subunit of rat liver DNA polymerase α on a two‐dimensional (2‐D) electrophoretic map. In this case a polyacrylamide gel containing DNA was used for the second dimension. DNA presence does not change the 2‐D protein pattern but makes it possible to conduct a polymerase reaction directly in the gel after separation. A crude extract of rat liver nuclei was used for analysis. The extract is quickly, isolated and contains mainly DNA polymerase α activity. It was shown that this enzyme restores its activity after 2‐D electrophoresis and sodium dodecyl sulfate (SDS) elution. After polymerase reaction with labeled dNTPs and autoradiography, the catalytic polypeptide or, rather, polypeptide cluster is revealed as chains of spots (possibly because of the presence of different hydrolyzed and phosphorylated forms). These spots are located on the 2‐D electrophoretic map in the region corresponding to molecular masses of 160, 140, and 130 kDa and pI 5.5–6.2.
The different expression of proteins recognized by monoclonal anti‐heat shock protein 70 (hsp70) antibody in human colonic diseases
Authors: Stul Jiřri , Bureš Jan , Jandík Pavel , Langr František , Kovářová Hana , Macela Aleš 
Published: May 2005

AbstractAnalysis of the expression of hsp70 (70 kDa heat‐shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high‐resolution two‐dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti‐hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1–5.3 range and in the molecular mass range of 68–69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.
Structural transitions of human serum albumin: An investigation using electrophoretic techniques
Authors: Gianazza Elisabetta , Galliano Monica , Miller Ingrid 
Published: May 2005

AbstractThe influence of pH and urea concentration on the electrophoretic mobility of native and reduced human serum albumin was evaluated by zonal electrophoresis across transverse urea gradients as well as by migration across transverse pH gradients in gels containing varying concentrations of urea. Exposure to urea results in a change of both pI and hydrodynamic volume of the albumin molecule. At acidic pH, the former effect is brought about by lower urea concentrations than the latter, as made evident by a biphasic denaturation curve; in alkaline buffers, all structural transitions occur at once, and a typical sigmoidal curve is observed. Below pH 6, the lower the pH, the lower the urea concentration causing albumin denaturation. For instance, in the presence of 3 M urea, below pH 5 > 95% of the protein is present in its denatured state, above pH 8 > 90% is in its native form, whereas in the 6.5–7.5 pH range the two components have similar abundance. Also, the reversibility of the transition between folding and unfolding depends upon pH, and is complete only above pH 6. After inclusion of β‐mercaptoethanol in the albumin sample the urea concentration required to bring about protein unfolding increases between pH 4 and 6 and decreases thereafter.
Prothymosin α: Efficient sequence determination by experimental and theoretical capillary electrophoretic support
Authors: Weiler Angelika , Stoeva Stanka , Tsitsilonis Ourania E. , Voelter Wolfgang 
Published: May 2005

AbstractA rapid and efficient primary structure elucidation of prothymosin (ProTα) and its enzymatic fragments was performed by a combination of matrix‐assisted laser desorption ionization mass spectrometry (MALDI), automatic N‐terminal Edman degradation, and the available theoretical predictions of electrophoretic mobility in capillary electrophoresis (CE) as a basis suggesting active sites of ProTα in the different bioassaysPresented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25; 1996
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Computer analysis of two‐dimensional electrophoresis gels: A new segmentation and modeling algorithm
Authors: Bettens Eva , Scheunders Paul , Van Dyck Dirk , Moens Luc , Van Osta Peter 
Published: May 2005

AbstractThe complexity of the spot patterns of two‐dimensional electrophoresis gels made it necessary to use image processing techniques to analyze the gels. An important issue in the analysis is the detection and quantification of the protein spots. In this paper we describe a new technique to segment and model the different spots. For the segmentation of a gel into its different spot regions we apply a watershed technique, which is robust and efficient. For the quantification of the spots, a new spot model is constructed, based on diffusion principles. Besides the advantage of having a physical interpretation, the model is demonstrated to be superior to the commonly used Gaussian models.