bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional electrophoretic analysis of human breast carcinoma proteins: Mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2
Authors: Rasmussen Richele K. , Ji Hong , Eddes James S. , Moritz Robert L. , Reid Gavin E. , Simpson Richard J. , Dorow Donna S. 
Published: May 2005

AbstractMLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701–710), comprises several distinct structural domains including an src homology‐3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N‐terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23–77), fused to glutathione S‐transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione‐Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2‐SH3 domain binding proteins from lysates of 35S‐labelled MDA‐MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low‐abundance proteins with a molecular weights (Mr) of ˜ 31 500 and ˜ 34 000, bound consistently to the MLK2N protein. To establish accurately the Mr / isoelectric point (pI) loci of these MLK2‐SH3 domain binding proteins, a number of abundant proteins in a two‐dimensional electrophoresis (2‐DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in‐gel proteolysis and fractionation by rapid (˜ 12 min)˜ microbore column (2.1 mm ID) reversed‐phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide‐mass fingerprinting and electrospray (ESI) – mass spectrometry (MS)‐MS utilizing capillary (0.2–0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2‐DE protein database for the human breast carcinoma cell line MDA‐MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: www.ludwig.edu.au/www/jpsl/jpslhome.html).
Length and sequence variation in D7S22 (g3) alleles studied by high resolution length measurements and nucleotide sequencing
Authors: Andreassen Rune , Olaisen Bjørnar 
Published: May 2005

AbstractIn a study of DNA sequence and length variation in the repeat array of small D7S22 alleles, 100 alleles typed as the common 14 repeat allele (14R) and 92 rare ones were selected for further characterization. A polymerase chain reaction (PCR) based allele length measurement method revealed a discontinuous distribution of alleles. The 92 rare alleles were grouped by their number of repeats. All, except four 6R alleles were distributed within the 11R–19R allele groups. The 14Rs revealed no further length variation while 7 out of the 92 rare alleles showed small length deviations from the other alleles within their respective groups. Nucleotide sequencing of the repeat array was performed in 17 alleles selected from each of the nine allele groups. The micro length variation within allele groups was caused by the presence of either 33, 36 or 37 bp repeats in given positions. A comparison of three 14Rs revealed no further sequence variation between these. Nine out of the fourteen repeats in the 14R differed in sequence and/or size. Based on this difference the repeat array sequence was converted into a code of different variant repeats. The 6R showed a variant repeat code quite unlike that of the 14R, while the encoded allele structure of the other rare alleles suggested that most of them may have evolved from a 14R allele by deletion or duplication of repeat units. Nucleotide sequencing of progenitor and mutant in a D7S22 de novo mutation as well as typing in a polymorphic site near the repeat array suggested that the event was an intra‐allelic deletion of exactly three repeats. The present findings indicate that the 14R is ancestral to most rare small alleles, and that mutations in small alleles most often are intra‐allelic events leading to a change in bp size equal to an integer number of repeats.
Capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary pH gradient
Authors: Stoyanov Alexander V. , Gelfi Cecilia , Righetti Pier Giorgio 
Published: May 2005

AbstractCapillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (β) at the pI (9.74) than the latter (pI 7.47), due to the favorable ΔpK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the β/λ ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest β/λ ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the β/λ ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5–10 Pharmalyte carrier ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the ‐OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam‐lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5–8.5 zone where, for Pharmalytes, the β/λ ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 μm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.
Capillary electrophoresis of glycosaminoglycan‐derived disaccharides: Application to stability studies of glycosaminoglycan chitosan complexes
Authors: Denuzière Anne , Taverna Myriam , Ferrier Danielle , Domard Alain 
Published: May 2005

AbstractCapillary zone electrophoresis (CZE) was used to separate the disaccharides produced by chondroitinase digestion of chondroitin sulfates. The main disaccharides formed upon depolymerization have identical charge and mass. Baseline resolution of these two compounds was achieved by selecting appropriate concentration and pH of a borate buffer. Validation of the method showed a good linearity of the response and a very satisfactory reproducibility of migration times with a relative standard deviation (RSD) of less than 0.4%. The reproducibility of peak areas was improved by using an internal standardization. The addition of cinnamic acid (internal standard) to the incubation medium allowed us to perform kinetic measurements of the depolymerization while keeping a baseline resolution of the two main disaccharides analyzed during the complete digestion course even when their concentration in the incubation medium increased. Application of this method to the comparison of the rate of hydrolysis of chondroitin sulfate and of a complex associating chondroitin sulfate with chitosan showed clearly that, at the physiological pH, chitosan protected the chondroitin sulfate from depolymerization. This phenomenon was more pronounced as the pH of the incubation medium was far from the optimum pH activity of the chondroitinase.
Sample matrix effects on glycopeptide stability by high performance capillary electrophoresis
Authors: Rush Robert S. , Boss Hollis J. , Katta Viswanatham , Rohde Michael F. 
Published: May 2005

AbstractHigh performance capillary electrophoresis (CE) of glycoprotein digests frequently reveals extensive microheterogeneity associated with specific protein glycosylation sites. The choice of the sample matrix can influence the electrophoretic migration time, peak shape and resolution, as well as the physical stability of the product glycopeptides. Acetic acid is a frequently employed sample matrix for both capillary electrophoresis and electrospray ionization‐mass spectrometry (ESI‐MS). Acetic acid appears to enhance the spontaneous hydrolysis of sialic acids from the nonreducing termini of glycopeptides in a time‐ and concentration‐dependent manner, even at 5°C, as evidenced by changes in the electrophoretic mobility and ESI‐MS spectra of the resulting glycopeptides. The observed parallel electrophoretic mobility changes for specific glycoforms are consistent with the induction of peptide structure with time. Asialoglycopeptide mobilities were stable in acetic acid. Electrophoretic mobilities can be stabilized with propionic acid sample matrix with no apparent structural changes observed by ESI‐MS within 31 h. Migration time reproducibility was in the range of 0.1% relative standard deviation (N = 7) with excellent peak shapes and enhanced glycopeptide resolution.
Characterization of thioredoxins by sodium dodecyl sulfate‐slab gel electrophoresis and high performance capillary electrophoresis
Authors: Bunik Victoria , Shoubnikova Alexandra , Bisswanger Hans , Follmann Hartmut 
Published: May 2005

AbstractDisulfide containing proteins – thioredoxins from E.coli and pig heart mitochondria – were characterized by sodium dodecyl sulfate (SDS)‐electrophoresis and high performance capillary electrophoresis (HPCE). Following the mitochondrial thioredoxin samples at different stages of purification, we found that their electrophoretic patterns vary, dependent on the redox condition of isolation, preparation of the samples for SDS‐electrophoresis, and sample storage. All these factors influenced the relative intensities of several protein bands with thioredoxin‐like mobility, whereas the sample storage also resulted in the appearance of SDS‐ and dithiothreitol (DTT)‐resistant high molecular mass forms, probably thioredoxin dimers. The multiple forms of the thioredoxin from pig heart mitochondria in SDS‐electrophoresis might be dependent on the oxidation state of the protein cysteine residues. A commercial preparation of the thioredoxin from E.coli did not exhibit any changes in mobility in SDS gels whether the sample was prepared with or without DTT. After the final purification step no correlation was found between mitochondrial thioredoxin activity, determined in the insulin assay, and its purity in SDS‐electrophoresis. A correlation was, however, found when analyzing the thioredoxin by HPCE. The latter approach demonstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active in the insulin assay. Also, thioredoxin from E.coli, homogeneous on SDS‐electrophoresis, was found heterogeneous on HPCE. The peak corresponding to the insulin‐dependent thioredoxin activity was split into two by DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCEPresented 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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Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis
Authors: Völkl Alfred , Mohr Heribert , Weber Gerhard , Fahimi H. Dariush 
Published: May 2005

AbstractRat hepatic peroxisomes (PO) were separated from other cell organelles by free flow electrophoresis (FFE) in combination with immunocomplexing PO prior to FFE with an antibody directed against the cytoplasmic aspect of the peroxisomal membrane protein PMP 70. This novel approach is based on a method termed antigen‐specific electrophoretic cell separation (ASECS) which was originally introduced for the isolation of human T and B lymphocyte subpopulations by Hansen and Hannig (J. Immunol. Methods 1982, 51, 197–208). We adapted this technique to PO isolation from a crude peroxisomal fraction, streamlining it by the following modifications: (i) The sandwich‐technique recommended to further lower a negative surface charge was renounced. (ii) Instead, the pH of the electrophoresis buffer was raised from 7.2 to 8.0, thus minimizing the electrophoretic mobility of the particles immunocomplexed due to the fact that the isoelectric point (pI) of IgG molecules is close to pH 8.0. PO isolated by this modification, referred to as immune free flow electrophoresis (IFFE), are as pure, intact, and structurally well‐preserved as are highly purified PO obtained by density gradient centrifugation. The technique is currently applied for the isolation of peroxisomal subpopulations that are difficult to obtain by means of density gradient centrifugationPresented 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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A new method to assign immunodetected spots in the complex two‐dimensional electrophoresis pattern
Authors: Zeindl‐Eberhart Evelyn , Jungblut Peter R. , Rabes Hartmut M. 
Published: May 2005

AbstractA new, easy method for the immunodetection of specific antigens in two‐dimensional electrophoresis (2‐DE) is described. Areas of 2‐DE gels containing antigens of interest are electrophoretically transferred to polyvinylidene difluoride membranes, immunostained with specific antibodies using Fast Red or 5‐bromo‐4‐chloro‐3‐indolyl phosphate/nitro blue tetrazolium as detection systems and counterstained with Coomassie Brilliant Blue. In contrast to conventional methods, it is possible to use this procedure to exactly assign immunoreacting proteins on a single blot to their corresponding and surrounding blue‐stained protein spots.
Three approaches to enantiomer separation of β‐adrenergic antagonists by capillary electrochromatography
Authors: Nilsson Staffan , Schweitz Leif , Petersson Maria 
Published: May 2005

AbstractThree different capillary electrochromatographic methods for the enantiomer separation of β‐adrenergic antagonists (acebutolol, alprenolol, atenolol, metoprolol, pindolol, prenalterol, and propranolol) were applied using different cyclodextrins (β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin and dimethyl‐β‐cyclodextrin) added to the electrolyte, a cross‐linked protein‐gel (cellobiohydrolase I) and a molecularly imprinted ((R)‐enantiomer of propranolol) superporous polymer as chiral selectors. Through use of these different separation strategies, all the β‐adrenergic antagonists studied could be resolved into their enantiomers, although the three methods were carried out without extensive optimization. The protein and molecularly imprinted phases gave the highest selectivities whereas employing cyclodextrins resulted in the highest separation efficiency. Proteins and cyclodextrins are primarily natural products, albeit the cyclodextrins can be derivatized. In contrast, the molecularly imprinted chiral stationary phase can be highly customized when produced.
Histamine‐modified β‐cyclodextrins for the enantiomeric separation of dansyl‐amino acids in capillary electrophoresis
Authors: Galaverna Gianni , Corradini Roberto , Dossena Arnaldo , Marchelli Rosangela , Vecchio Graziella 
Published: May 2005

AbstractTwo novel monosubstituted β‐cyclodextrins (CD) bearing the histamine moiety linked to the upper CD rim, either through the amino group or the imidazole nitrogen 1N, CD‐hm and CD‐mh, were successfully used as chiral selectors in capillary electrophoresis for the enantiomeric discrimination of dansyl (Dns)‐amino acids. Good results were obtained by using low concentrations of the selectors (1–3 mM). The effect of pH on the chiral discrimination was studied in order to modulate the number and the position of the positive charges. By increasing the pH from 5 to 7.5, chiral discrimination decreased along with the deprotonation of the imidazolyl moiety. Inversion of the migration order was observed with the two CDs, depending on the relative position of the charged moieties on the upper rim. Ion pair interaction coupled to inclusion complexation seems to account for the discrimination process. The effects of the temperature, CD concentration and capillary length on chiral resolution were also examined.
Separation of synthesized enantiomers and diastereomers of thiino‐[4,3‐b]pyrrole‐2,3‐dione derivatives and 2H‐thiopyrans by capillary electrophoresis
Authors: Blitzke Thorsten , Wilde Horst , Vogt Carla 
Published: May 2005

AbstractCapillary electrophoresis was used to separate diastereomers and enantiomers of synthesized thiopyrans and thiinopyrroles. Separation conditions were optimized by varying the buffer parameters, i.e., kind and concentration of chiral selector, pH, main buffering component, micelle forming additives, and content of organic solvents. The interaction of thiopyrans and thiinopyrroles with β‐ and γ‐cyclodextrins results in the separation of enantiomers and diastereomers. The magnitude of the resolution depends on the spatial requirements of the different substituents of the analytes and the dimensions of the cyclodextrin cavity. For well‐separated enantiomers or diastereomers the identification and characterization of degradation products was possible.
Enantioselective determination of 3,4‐methylene‐dioxymethamphetamine and two of its metabolites in human urine by cyclodextrin‐modified capillary zone electrophoresis
Authors: Lanz Matthias , Brenneisen Rudolf , Thormann Wolfgang 
Published: May 2005

AbstractUsing capillary zone electrophoresis with a phosphate buffer at pH 2.5 containing 30 mM (2‐hydroxypropyl)‐β‐cyclodextrin as chiral selector, the simultaneous separation of the enantiomers of 3,4‐methylenedioxymethamphetamine (MDMA or Ecstasy) and its two metabolites 4‐hydroxy‐3‐methoxymethamphetamine (HMMA) and 3,4‐methylenedioxyamphetamine (MDA) in human urine is reported. The assay described is based upon enzymatic hydrolysis of conjugated HMMA (major urinary metabolite) and solid‐phase extraction followed by injection of a few nL of the extract onto a 50 μm internal diameter (ID) fused‐silica capillary of 60 cm length. Solutes are detected via on‐column absorbance at 195 nm. For 375 ng/mL drug levels, intraday and interday imprecision is < 4%. With 5 mL urine samples, the detection limit is in the 20–50 ng/mL range. Via analysis of the urines of two patients, the metabolism of MDMA is demonstrated to be enantioselective, with significantly higher urinary amounts of R‐(−)‐MDMA being excreted compared to S‐(+)‐MDMA. Within 72h after drug administration one patient was determined to excrete 42.28 and 10.16% of the racemic MDMA dose (1.5 mg/kg body weight) as R‐(−) and S‐(+)‐MDMA enantiomers, respectively. Corresponding values for the second subject were found to be 28.63 and 9.34%. The metabolism of the enantiomers of the two metabolites showed interindividual differences. The first and second detected HMMA enantiomers represented 3.79 and 5.42% (first subject) and 8.51 and 4.36% (second), respectively, of the administered MDMA dose. For the MDA enantiomers, corresponding values were 2.44, 1.76, 0.75, and 0.79%, respectively.
A role for Edman degradation in proteome studies
Authors: Gooley Andrew A. , Ou Keli , Russell Jim , Wilkins Marc R. , Sanchez Jean‐Charles , Hochstrasser Denis F. , Williams Keith L. 
Published: May 2005

AbstractAdvances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two‐dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottle‐neck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N‐terminal sequence tag.
Models of mobility‐shift assay of complexes between dimerizing protein and DNA
Authors: Cann John R. 
Published: May 2005

AbstractThe theory of mass transport coupled to macromolecular interactions under chemical kinetic control forms the basis of four different models of the electrophoretic mobility‐shift assay of complexes formed between dimerizing proteins and DNA. The theory of mass action was applied to the set of simultaneous dimerization (either simple or ligand‐induced) and DNA‐binding reactions in order to fix the initial equilibrium composition of mixtures to be assayed. Theoretical mobility‐shift patterns were obtained for a range of protein concentrations at constant DNA concentration by numerical solution of the set of simultaneous transport‐reaction equations appropriate for each model. In those cases in which dimerization in solution is modeled (including heterodimerization), analysis of the peaks in the patterns provides apparent binding constants, which, when extrapolated to infinite dilution of protein, yield acceptable estimates of equilibrium constants. Those for binding of dimer are products of two or three equilibrium constants, from which the equilibrium binding constant can be extracted, privided that dimerization and, where required, ligand‐binding constants are determined by independent physicochemical methods. Dimerization of protein when bound to DNA is distinctive in that extrapolation to infinite dilution of protein is not required.