bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping of Chlamydia trachomatis proteins by Immobiline‐polyacrylamide two‐dimensional electrophoresis: Spot identification by N‐terminal sequencing and immunoblotting
Authors: Bini Luca , Sanchez‐Campillo Maria , Santucci Annalisa , Magi Barbara , Marzocchi Barbara , Comanducci Maurizio , Christiansen Gunna , Birkelund Sevend , Cevenini Roberto , Vretou Evangelia , Ratti Giulio , Pallini Vitaliano 
Published: May 2005

AbstractProteins from purified elementary bodies of Chlamydia trachomatis were separated by two‐dimensional gel electrophoresis on nonlinear wide‐range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N‐terminal amino acid sequencing, we established the positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein‐rich outer membrane protein (OMP2), the DnaK‐like, GroEL‐like, and macrophage infectivity potentiator (MIP)‐like proteins, the plasmid‐encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein‐elongation factor EF‐Tu. Other proteins, for which gene assignment was not possible, have been identified by three parameters (Mr, pI and N‐terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome‐linked database of chlamydial proteins.
Use of internally controlled reverse transcriptase‐polymerase chain reaction for absolute quantitation of individual multidrug resistant gene transcripts in tissue samples
Authors: Zhang Fang , Riley Joan , Gant Timothy W. 
Published: May 2005

AbstractResearch into the relative importance of P‐glycoprotein (pgp) overexpression, in comparison to other mechanisms of multidrug resistance (mdr) phenotype development, has been hampered by difficulties of measurement in clinical samples. The small size, heterogeneity and often poor quality of clinical specimens renders the quantitation of mdr mRNA species by Northern analysis or RNAse protection difficult. The reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay has both the sensitivity and specificity to make it suitable for the analysis of mdr mRNA levels in clinical samples. It also has a significant speed advantage over Northern and RNAse protection analysis. Unfortunately the variable nature of the reactions involved have made it difficult to obtain accurate and reproducible quantitative data. To overcome this difficulty we constructed internal RNA standards to each human and rat mdr mRNA species. When included in the RT‐PCR reaction these internal standards allow both comparative and absolute quantitation of individual mdr family mRNA levels.
Detection of glycosaminoglycans as a copper (II) complex in capillary electrophoresis
Authors: Toida Toshihiko , Linhardt Robert J. 
Published: May 2005

AbstractGlycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronic acid were analyzed by reversed polarity capillary electrophoresis. Detection was achieved at 240 nm based on the formation of a copper (II) complex in copper sulfate solution at low pH. Glycosaminoglycans having a high ratio of iduronic acid to glucuronic acid, as well as ones having lower molecular weight, gave the highest detection sensitivity using this method. Detection of the copper (II)‐heparin complex is extremely sensitive, permitting the analysis of as little as 10−9 g. This method was also successfully applied to the analysis of heparin oligosaccharides that lacked a chromophore prepared from heparin using controlled, low pH nitrous acid depolymerization.
Acid‐catalyzed reductive amination of aldoses with 8‐aminopyrene‐1,3,6‐trisulfonate
Authors: Evangelista Ramon A. , Guttman András , Chen Fu‐Tai A. 
Published: May 2005

AbstractThe reductive amination of monosaccharides with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS) in seven different organic acids including the commonly used acetic acid was investigated by capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection. The correlation between the yields of the saccharide‐APTS adducts and pKa of the organic acid catalyst is consistent with general acid catalysis of the rate‐determining step of the reductive amination reaction. Derivatization in the presence of organic acids of higher strength than acetic acid produced substantially higher yields of APTS‐sugar adducts, an effect which is more pronounced for N‐acetylamino sugars. Optimum yields were obtained using citric acid as a catalyst. Conversion of a few nanomoles of neutral saccharides to the APTS derivatives is achieved at 75°C in less than 60 min.
Determination of carbohydrates as their dansylhydrazine derivatives by capillary electrophoresis with laser‐induced fluorescence detection
Authors: Perez Sherryl A. , Colón Luis A. 
Published: May 2005

AbstractA mixture of mono‐ and disaccharides was derivatized with dansylhydrazine in a relatively fast chemical reaction (15 min) which is selective toward aldehydes and ketones. The products of the derivatization reaction were then separated by capillary electrophoresis (CE) using laser‐induced fluorescence (LIF) as the detection mode. The smallest amount of carbohydrate derivatized and determined by CE/LIF was 10 pmol contained in 100 μL of the final reaction mixture. The CE/LIF method provided a linear response for the carbohydrates tested (over 2–3 orders of magnitude), with a limit of detection of 100 amol. Separation efficiencies over 250 000 theoretical plates were obtained. The procedure was used to analyze a tear fluid sample to demonstrate the applicability of the method for the glucose determination in small volumes of biological samples.
Analysis of Stevia glycosides by capillary electrophoresis
Authors: Mauri Pierluigi , Catalano Giovanna , Gardana Claudio , Pietta Piergiorgio 
Published: May 2005

AbstractThe determination of diterpene glycosides from Stevia rebaudiana leaves using capillary electrophoresis is described. Analyses were performed on fused silica capillaries with 20 mM sodium tetraborate buffer, pH 8.3, and 30 mM sodium dodecyl sulfate. The effect of the organic solvent injected with the sample solution on the electrophoretic solution has been confirmed, and an absolute amount of 1.6 nL per injected sample was optimal. Rebaudioside A and steviolbioside were isolated by semipreparative high performance liquid chromatography (HPLC), and their structure was assessed by mass spectrometry.
Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent‐labeled substrate
Authors: Zhang Zhiquan , Pierce Margaret L. , Mort Andrew J. 
Published: May 2005

AbstractA sensitive assay is described for the detection of pectate‐depolymerizing enzymes using capillary electrophoresis of a fluorescent end‐labeled pectate oligomer. The labeled oligomer is allowed to react with the enzyme either in vitro or in vivo, such as inside the intercellular spaces of a cotton cotyledon, and after an appropriate incubation time the products are analyzed by capillary electrophoresis. The site and mode of action of the pectate‐depolymerizing activity can be inferred from the products. Both endo‐ and exopolygalacturonase activity, and lyase activity, were distinguished. Since only the fluorescent oligomer and products from its labeled reducing end are detected, there is no interference from other compounds; only pectic enzyme activity is detected. By this type of analysis we can show that there is considerable endo‐ and exopolygalacturonase activity in the intercellular spaces of cotton cotyledons.
Immobilized metal ion affinity gel electrophoresis: Quantification of protein affinity to transition metal chelates
Authors: Baek Won‐Ok , Haupt Karsten , Colin Carole , Vijayalakshmi Mookambeswaran A. 
Published: May 2005

AbstractThis paper describes some recent advances in the methodology of immobilized metal ion affinity gel electrophoresis. Four different ways to incorporate metal chelate ligands in agarose and polyacrylamide‐based electrophoresis gels are evaluated, a new polymerizable metal chelating ligand, allyl–2‐hydroxy‐3‐(N,N‐dicarboxymethyl)amino‐propyl ether, is introduced, and the determination of affinity constants described. The affinities of model proteins (ribonucleases A and B and cytochromes c from different species) for the transition metal chelate iminodiacetic acid‐Cu(II) were studied. The results were found to be in agreement with literature data on immobilized metal ion affinity chromatography, and the polymer nature and the different chemistries used influenced the affinity only quantitatively, keeping the basic mechanisms of interaction unchanged.
Federated two‐dimensional electrophoresis database: A simple means of publishing two‐dimensional electrophoresis data
Authors: Appel Ron D. , Barioch Amos , Sanchez Jean‐Charles , Vargas J. Reynaldo , Golaz Olivier , Pasquali Christian , Hochstrasser Denis F. 
Published: May 2005

AbstractWhile a two‐dimensional electrophoresis (2‐DE) database is a relatively old concept, in recent years it generated renewed interest within the 2‐DE community due to two main factors: (i) The high reproducibility of the current 2‐DE method allows 2‐DE images to be exchanged and compared between laboratories. (ii) The recent development of faster and more powerful techniques for protein identification such as microsequencing, matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) and amino acid composition makes the production of reference protein maps and 2‐DE databases cost‐ and time‐effective. Additionally, the Internet network's current increase in popularity, combined with the rapid growth of Internet‐connected laboratories, provides a straightforward means of publishing and sharing 2‐DE data. While a small number of laboratories have already successfully published their data over the net, the increasing number of 2‐DE database servers that are currently being set up will sooner or later require some kind of standardization. Unfortunately, standardization can be a long and cumbersome process inevitably leading to undesirable compromises. A federated database offers a simple and efficient way to publish and share 2‐DE data without the need for standardization. Taking advantage of Internet protocols such as World Wide Web, they allow each laboratory to maintain their own database and to interconnect it with other similar databases through the use of active cross‐references. This paper first presents guidelines for building a federated 2‐DE database that may easily be followed by most laboratories. It then briefly reviews the state‐of‐the‐art in networked 2‐DE databases, and finally describes the SWISS‐2DPAGE database which fully implements the concept of a federated 2‐DE database.
The yeast SWISS‐2DPAGE database
Authors: Sanchez Jean‐Charles , Golaz Olivier , Frutiger Séverine , Schaller Dominique , Appel Ron D. , Bairoch Amos , Hughes Graham J. , Hochstrasser Denis F. 
Published: May 2005

AbstractThe systematic sequencing of the yeast genome will soon be completed. A new challenge has been launched by the EUROFAN (European Functional Analysis) project whose goal is to elucidate the physiological and biochemical function of newly discovered open reading frames (ORF) from yeast. One of the approaches is to use protein‐based technologies such as two‐dimensional gel eletrophoresis and protein identification in order to establish a yeast reference map. Modified protein patterns can be compared to the reference map which hopefully will help identify changes related, for example, to growth processes or developmental events. This paper describes the yeast SWISS‐2DPAGE database in which charge separation was obtained using immobilized pH gradient (IPG). Proteins identified by gel comparison, amino acid composition analysis and/or microsequencing are recorded and described in an accessible uniform format. We have identified more than one hundred polypeptides, several of which were newly mapped. In addition, the yeast SWISS‐2DPAGE database can be freely accessed through the World Wide Web (WWW) network on the ExPASy molecular biology server.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and centrifuge blotting: Preparation of polypeptides for amino‐terminal sequence analysis
Authors: Hermansen Leonila F. , Juul Jessie , Sletten Knut 
Published: May 2005

AbstractThe applicability and reproducibility of a previously described (L. F. Hermansen et al., Electrophoresis 1993, 14, 1302–1306) centrifuge‐blotting procedure for capturing subnanomolar amounts of protein on polyvinylidene difluoride membranes for direct Edman degradation was further investigated. Proteins with different molecular masses were centrifuge‐blotted onto Immobilon CD membranes. Simultaneous blotting and desalting was achieved with an overall yield of 15–56% after 2 h centrifugation for proteins with a molecular mass of 12–30 kDa. Centrifugation of myoglobin for 6 h resulted in an overall yield of 72%. The subnanomolar amounts obtained were also sufficient to conduct cyanogen bromide cleavage in situ on proteins with blocked NH2‐terminus and to generate sequence information.
Capillary zone electrophoresis in agarose gels using absorption imaging detection
Authors: Palm Anders , Lindh Curt , Hjertén Stellan , Pawliszyn Janusz 
Published: May 2005

AbstractA simple home‐built electrophoretic unit and a commercially available chargecoupled device (CCD) camera with image acquisition and analysis software were used to study the separation process in zone electrophoresis experiments in 4 cm long, round capillaries (inside diameter 0.2 mm). Several capillaries could be investigated simultaneously. The absorption imaging system was used not only to follow the course of the separation but also to study the interaction between biologically active substances (proteins, detergents, enzymes and substrate). Since the system allows visual on‐line observation of the separation one can rapidly decide when the analysis is finished, which often shortens the analysis time. The electrophoresis method presented is suitable also for preparative runs, since direct visualizations of a solute zone allows it to be excised and then used for further studies.
Alpha‐2‐HS‐glycoprotein (AHSG) polymorphism in semen and saliva
Authors: Yasuda Toshihiro , Takeshita Haruo , Tsubota Etsuko , Sawazaki Kazumi , Iida Reiko , Nadano Daita , Kishi Koichiro 
Published: May 2005

AbstractPolymorphism of alpha‐2‐HS‐glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin‐layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P–40 into the IEF gel (pH 4.2–4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2–5 μL semen and 50–100 μL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.
Identification of stress proteins in endothelial cells
Authors: Portig Irene , Pankuweit Sabine , Lottspeich Friedrich , Maisch Bernhard 
Published: May 2005

AbstractEndothelial cells have been shown to play a major role in the pathophysiology of various diseases including ischemic heart disease and viral infection leading to myocarditis or dilated cardiomyopathy, conditions in which stress proteins (heat shock protein – hsp; glucose‐related protein – grp) are likely to be involved. For further characterization of stress proteins and their possible role in these diseases, the major stress proteins in human endothelial cells were separated by two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension and identified by immunoblotting and either N‐terminal or internal amino acid sequencing, respectively. Ubiquitin, hsp27, hsp60, hsp70, heat shock cognate protein 70, grp78 and grp75 were found to be constitutively expressed; hsp72 was found in stressed cells, exclusively, in line with results obtained in other human cell lines. Three additional proteins with molecular masses between 34 and 40 were regulary detected in stressed cells that were found to have identical amino acid sequences with those of members of the hsp70 familiy.
S‐Pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine‐containing peptides
Authors: Moritz Robert L. , Eddes James S. , Reid Gavin E. , Simpson Richard J. 
Published: May 2005

AbstractIn‐gel proteolytic digestion of acrylamide‐gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide‐mass fingerprinting. However, it is well recognised for disulfide‐bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in‐gel protein digestion. It consists of first reducing and S‐pyridylethylating Coomassie Brilliant Blue R‐250‐stained proteins immobilised in the whole gel slab with dithiothreitol and 4‐vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (≤12 min) reversed‐phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S‐pyridylethylation of acrylamide‐immbilised proteins prior to in‐gel digestion. Further, the levels of gel‐related contaminants, which otherwise result in suppression of sample signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction / alkylation step. Additionally, we demonstrate that S‐β‐(4‐pyridylethyl)‐cysteine containing peptides can be readily identified during reversed‐phase HPLC by absorance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic‐pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S‐β‐(4‐pyridylethyl)‐cysteine during Edman degradation, and by tandem mass spectrometry.
Ultraviolet matrix assisted laser desorption ionization‐mass spectrometry of electroblotted proteins
Authors: Schreiner Martin , Strupat Kerstin , Lottspeich Friedrich , Eckerskorn Christoph 
Published: May 2005

AbstractDirect mass spectrometric analysis of proteins electroblotted onto polyvinylidene fluoride membranes after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis is demonstrated by matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) with a linear time‐of‐flight instrument, equipped with a nitrogen laser (337 nm). The blotted proteins were desorbed directly from the blotting membrane after incubation with sinapinic acid as matrix. Different commercially available membranes resulted in high quality protein signals for hydrophobic membranes exhibiting high specific surface areas (Immobilon PSQ or Trans‐Blot) or for charged membranes (Immobilon CD). Systematic investigations with standard proteins were performed to compare standard preparation procedures for ultraviolet (UV) MALDI‐MS on stainless steel sample stages and preparation of proteins immobilized onto membranes either by direct application from protein solutions (spotting) or by electrotransfer from gels (electroblotting). Aspects such as mass resolution, reproducibility from shot to shot and spot to spot, mass accuracy, and preservation of protein localization are addressed in this paper.
Statistical features in the lakes‐straits model and the influence of hernias
Authors: Disch Christian , Loomans Dirk , Sokolov Igor M. , Blumen Alexander 
Published: May 2005

AbstractWe evaluate numerically the mobility of DNA chains under field‐inversion gel electrophoresis (FIGE) conditions in the framework of the lakes‐straits model introduced by Zimm (Phys. Rev. Lett. 1988, 61, 2965–2968; J. Phys. Chem. 1991, 94, 2197–2206). We extend the model by allowing both simple and also multiple‐branched hernias; this is achieved by arranging the data structure used in the algorithm so that each fragment in a lake can be treated separately. We show that the existence of hernias allows the probe to migrate faster and that with hernias the mobility minimum in FIGE shifts to smaller field periods. These effects occur only if the electric field is strong enough. We also discuss the influence of the model's parameters on the mobility.
Automatic matrix determination in four dye fluorescence‐based DNA sequencing
Authors: Yin Zhongbin , Severin Jessica , Giddings Michael C. , Huang Wei‐an , Westphall Michael S. , Smith Lloyd M. 
Published: May 2005

AbstractThe four dye fluorescence detection strategy is a widely used approach to automated DNA sequence analysis. An important aspect of data processing in this approach is the multicomponent analysis to deduce the concentrations of four fluorophores from fluorescence emission intensities at four different wave‐lengths. This requires knowledge of the correct transformation matrix M. The matrix M is a function both of the fluorophores employed and the fluorescence detection system. M is typically determined either by a calibration process with individual dyes, or by choosing four well‐separated individual peaks corresponding to the four different dyes. Both are time‐consuming and complicated procedures for routine use. An automatic scheme for finding M directly from raw sequence data is presented here. This facilitates data analysis and the underlying algorithm may also find utility in other multispectral applications.
Gel and free solution electrophoresis of variably charged polymers
Authors: Hoagland David A. , Smisek David L. , Chen Derek Y. 
Published: May 2005

AbstractTo assess the role of charge density on polyelectrolyte mobility, both gel and free solution electrophoresis experiments are performed on poly(acrylic acid) and acrylic acid/acrylamide copolymers. Control of charge density for poly(acrylic acid) is achieved through solution pH, while control for acrylic acid/acrylamide copolymers is obtained through chain composition. In either approach, the effective fraction of charged repeat units can be varied from 0 to 100% without a major interruption of solvent quality. Polyelectrolyte mobility in the presence of a monovalent counterion is observed to rise linearly with charge density when this density is low. A transition to charge density independence then occurs over a surprisingly narrow window of charge density. For vinyl polymers of the sort examined here, the transition occurs when 35–40% of the repeat units are charged. These observations are qualitatively consistent with the free solution electrophoresis model proposed by Manning and several previous data sets. An unexpected overlap of normalized gel and free solution data reveals that the charge density exerts a comparable influence in either environment. Results from the present study help define the experimental conditions in which electrophoresis can provide polymer separation by charge density and those in which the method can provide polymer separation by molecular weight.