bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis
Authors: Michaud Dominique , Cantin Line , Bonadé‐Bottino Michel , Jouanin Lise , Vrain Thierry C. 
Published: May 2005

AbstractThe biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla. M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second‐stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans‐epoxysuccinyl‐L‐leucylamido‐(4‐guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class‐specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition by OCI was partly prevented during electrophoresis. This species‐related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne.
Amino acid analysis of proteins separated by two‐dimensional electrophoresis in maize: Isoform detection and function identification
Authors: Touzet Pascal , de Vienne Dominique , Huet Jean‐Claude , Ouali Christian , Bouet Françoise , Zivy Michel 
Published: May 2005

AbstractThe possibility of using experimentally determined amino acid composition to assess relatedness between 75 proteins separated by two‐dimensional electrophoresis (2‐DE) and to identify them was tested on maize. Two independent parameters, the relative Euclidean distance and the correlation coefficient between the amino acid compositions, were evaluated and used. Previous sequence information for 31 out of the 75 proteins made it possible to evaluate the method for the detection of isoforms and for identification. However, the extension of the interrogation beyond maize to all plant sequences raised the problem of false positives that could nevertheless be limited by replications and by using additional information. The efficiency of the method to assess relatedness betwen proteins should make amino acid composition analysis a valuable tool in large protein characterization programs based on 2‐DE, by facilitating the transfer of information from one well‐documented organ/tissue or genotype to another.
High‐resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy‐transfer fluorescent primers
Authors: Wang Yiwen , Wallin Jeanette M. , Ju Jingyue , Sensabaugh George F. , Mathies Richard A. 
Published: May 2005

AbstractShort tandem repeat regions (STRs) from the polymorphic loci VWFA, THO1, TPO and CSF were amplified by the multiplex polymerase chain reaction (PCR) and analyzed by capillary array electrophoresis with fluorescence detection of energy transfer (ET) labels. The fluorescent ET primers are labeled with one fluorescein at the 5′ end and a second fluorescein at the position of the 7th or 9th (modified) base to produce fragments that fluoresce in the green (λmax = 525 nm). M13 A‐track sequencing fragments, used as an internal sizing standard, were generated with a universal primer that has a donor fluorescein at the 5′ end and a rhodamine acceptor at the position of the 11th (modified) base to produce fragments fluorescing in the red (> 590 nm). The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two‐color confocal fluorescence scanner. Separations were performed on arrays of hollow fused silica capillaries filled with denaturing and replaceable hydroxyethyl cellulose sieving matrices. Separations were complete in less than 50 min, and single base resolution as well as reproducible STR sizing was achieved. The relative standard deviation in sizing was below 0.6%. This work establishes the feasibility of high‐resolution, high‐speed and high‐throughput STR typing of single‐stranded DNA fragments using capillary array electrophoresis.
Characterization of mitochondrial DNA using lowstringency single specific primer amplification analyzed by laser induced fluorescence – capillary electrophoresis
Authors: Marino Michael A. , Weaver Kristal R. , Tully Lois A. , Girard James E. , Belgrader Phillip 
Published: May 2005

AbstractPolymerase chain reaction (PCR)‐based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low‐stringency sequence‐specific PCR (LSSP‐PCR) is an example of a recent method that does not require additional biochemical treatments. The analysis of LSSP‐PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D‐loop region is described. The DNA from five individuals were amplified (first step) using sequence‐specific primers to produce 1021 bp fragments containing the D‐loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO‐1 and laser‐induced fluorescence (LIF) detection. The LSSP‐PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.
Analysis of RNA by capillary electrophoresis
Authors: Skeidsvoll Jarle , Ueland Per Magne 
Published: May 2005

AbstractAnalytical parameters known to be important for the separation of DNA by capillary electrophoresis, including gel polymer concentration, electrical field strength and temperature, were investigated and optimized for the analysis of RNA molecules from 100 to 2000 bases. Denaturation, essential to obtain uniform and identifiable peaks, was accomplished by heating the sample in 80% formamide prior to electrophoresis and the presence of 2–8 M urea in the electrophoresis buffer. Efficient separations were obtained over a wide range of electrical field strengths and temperatures using the gel polymer hydroxypropylmethylcellulose (HPMC) as separation matrix. Low HPMC concentrations (< 0.3%) were suited for the separation of high molecular mass RNA (> 1000 bases) whereas higher HPMC concentrations were required for optimal separation of low molecular mass RNA. An optimized system was applicable for the separation of the predominating RNA populations (small RNA of 60–300 bases (as a group of unseparated peaks), 18S and 28S rRNA) in total RNA from a human glioma cell line. This is the first systematic investigation of electrophoresis of higher molecular mass RNA in capillaries, and motivates further studies to transfer electrophoresis of RNA to the capillary format.
Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA
Authors: Valentini Alessio , Timperio Anna Maria , Cappuccio Irene , Zolla Lello 
Published: May 2005

AbstractThe random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.
A procedure for protein elution from reverse‐stained polyacrylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis
Authors: Castellanos‐Serra Lila R. , Fernandez‐Patron Carlos , Hardy Eugenio , Huerta Vivian 
Published: May 2005

AbstractWe developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent‐free buffer (elution time 2 × 10 min, total working time about 30 min) with high yields (90–98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields. For N‐terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean‐up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N‐terminal sequenced.
Analysis and general classification of serum cryoglobulins by capillary zone electrophoresis
Authors: Shihabi Zakariya K. 
Published: May 2005

AbstractThe analysis and general classification of serum cryoglobulins (CG) by capillary zone electrophoresis (CZE) are described. This technique allowed the introduction of several new aspects to the analysis of CG: (i) direct detection of monoclonality of the γ‐globulins and general classification of the different CG types, (ii) a tenfold decrease in sample size, (iii) direct quantification of the cryoprecipitate without the need for further chemical analysis, and (iv) mathematical correction for the coprecipitation to avoid extensive washing steps or falsely elevated results. In addition to that, since staining steps are avoided, the method is rapid and easy to perform. The method has the limitation of not being able to perform immunological characterization directly of the CG. One hundred and ninety‐eight samples were analyzed by this technique. Many of the cryoprecipitates have a higher ratio of γ‐globulins/albumin when analyzed by CZE compared to analysis by agarose electrophoresis. Clinical examples are presented to illustrate the advantages of this method for the analysis of serum cryoglobulins.
A taxonomic study of the genus Listeria using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of intracellular proteins
Authors: Finn Brian , Rowe Michael 
Published: May 2005

AbstractFifty‐one strains of Listeria spp. and one strain of Jonesia denitrificans were subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and the resulting histograms compared using the unweighted pair group method with averaging. The majority of L. monocytogenes strains formed a discrete cluster with a minimum correlation of 73% similarity. The position of strain NCTC 11994 within the main L. monocytogenes cluster supports the recommendation by the National Collection of Type Cultures that it, rather than NCTC 10357, be adopted as the type strain of the species. All L. innocua strains formed a discrete cluster with a minimum correlation of 85% similarity. The similarity shown by L. grayi and L. murrayi, both between each other and with the other Listeria strains tested, support their designation as a single species within the genus Listeria. Although the single strain of J. denitrificans showed the least similarity with the other strains it was not sufficient to warrant support for its removal from the genus Listeria. This limited study provides a different perspective both between species in the genus Listeria and between the genera Listeria and JonesiaPresented at the 25th meeting of the British Electrophoresis Society, Maidstone, UK, July 13–14, 1995.
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Use of wide‐bore capillaries in constant denaturant capillary electrophoresis
Authors: Li Ziao‐Cheng , Thilly William G. 
Published: May 2005

AbstractA serious limitation of capillary electrophoresis in separating mutant from wild‐type DNA sequences is the amount of DNA which may be loaded on narrow‐bore columns. Because we sought to examine mutant sequences derived from more than 109 human cells, we were obliged to overcome this limitation. Thus we examined the feasibility of using wide‐bore capilllaries of 250, 320 and 540 μm in constant denaturant capillary electrophoresis (CDCE) using conditions which successfully limited the effect of Joule heating. Excellent separations were observed using the wide‐bore capillaries comparable to those obtained using a 75 μm capillary. DNA loading capacities were increased almost 100‐fold as the bore of capillary increased from 75 to 540 μm.
Separation of aracytidine and cytidine by capillary electrophoretic techniques
Authors: Křivánková Ludmila , Košťálová Andrea , Vargas Gabriela , Havel Josef , Boček Petr 
Published: May 2005

AbstractAracytidine (cytarabine, 1‐β‐D‐arabinofuranosylcytosine) is a synthetic analog of cytidine in which ribose is substituted by arabinose; it is used as a drug for the treatment of leukemia. A fast and reliable capillary electrophoretic method for the analysis of cytarabine and cytidine is described. The procedure utilizes the interactions with sodium dodecyl sulfate (SDS) micelles and borate, present in the background electrolyte, for the mobilization and selective separation of the analytes. The detection is carried out by UV absorbance at 275 nm. The method was applied both to pharmaceutical preparations and human serum. Analysis of an untreated serum requires 15 min; the detection limit is 0.8 μg/mL and the relative standard deviation (RSD) is 5.3%.
A method to determine the filter matrix in four‐dye fluorescence‐based DNA sequencing
Authors: Huang Weian , Yin Zhongbin , States David J. , Thomas Lewis J. , Fuhrmann Daniel R. 
Published: May 2005

AbstractIn a previous paper (Yin et al., Electrophoresis 1996, 17, 1143–1150), an automated method for matrix determination in four‐dye fluorescence‐based DNA sequencing was presented. As a continuation of that work, we have developed an alternative method to estimate the matrix from raw sequence data. The method uses an iterative clustering technique to associate each 4 × 1 data vector with one column of the desired filter matrix, using Kullback's I‐divergence as a distance measure. The method requires less preprocessing of the data and less computation than the approach described by Yin et al. (Electrophoresis 1996, 17, 1143–1150). An example demonstrating applicability of the proposed method to Applied Biosystems sequencer data is given.
Resolution of high molecular weight proteins in dependence on electric field strength in polyacrylamide gel electrophoresis
Authors: Starita‐Geribaldi Mireille , Houri André 
Published: May 2005

AbstractResolution of high molecular weight proteins, in the upper region of polyacrylamide gels, was studied in relation to the type of electric field. Separations by constant field gel electrophoresis (CFGE) were compared to those in pulsed oscillatory high‐performance electrophoresis (POPE), a novel technique which allows electrophoresis at high field strengths owing to a novel local field distribution. This distribution contributes to structural and mechanical stability of the gel with resultant well‐reproducible separation, enhanced resolution, and higher absolute mobility of proteins in POPE.
Size‐based separation of polyelectrolytes by capillary zone electrophoresis: Migration regimes and selectivity of poly(styrenesulphonates) in solutions of derivatized cellulose
Authors: Minárik Marek , Gǎs Bohuslav , Kenndler Ernst 
Published: May 2005

AbstractThe migration regimes and the separation selectivity of linear poly(styrenesulfonates) (PSS) were investigated in solutions of linear polymeric hydroxyethylcellulose (HEC). Variable parameters were the number of PSS monomer units (in the range of 25–6520), HEC concentration (below and above the entanglement threshold), molecular mass of HEC (between 35900 and 438000 Da) and electric field strength (from 190 to 1140 V/cm). Besides those regimes which are known for migration of polyelectrolytes in physical networks (Ogston sieving, reptation without and with orientation), the observed separation below the entanglement threshold indicates an effect of solute‐chain interactions similar to that observed for the separation of double‐stranded DNA. From the dependence of the separation selectivity on HEC concentration and molecular weight it was found that both parameters increase the separation. The selectivity of the solutions of higher concentrated low molecular HEC was compared to lower concentrated high molecular HEC, which has a similar bulk viscosity, and therefore obeys comparable peak dispersion due to thermal broadening. At high field strengths (preferably used in capillary electrophoresis) the former gave better separation only for shorter PSS molecules. For separation of longer PSS molecules, only high molecular HEC is applicable due to the deformation of the low molecular matrix by large analyte molecules.
Apolipoprotein AI isoforms in serum determined by isoelectric focusing and immunoblotting
Authors: Contiero Eva , Ferrari Roberto , Vaselli Giorgina Maria , Folin Marcella 
Published: May 2005

AbstractQuantitative evaluation of serum apolipoprotein AI (apo AI) isoforms through densitometric analysis following isoelectric focusing (IEF) is described. The apo AI isoforms were identified by immunoblotting. By combining these techniques, a qualitatively invariant pattern was observed in 54 serum samples, obtained from 27 men and 27 women. Peak 1 (proapo AI, pI 5.75) accounts for about 8% of the total densitometric area, peak 3 (apo AI0, pI 5.59) for about 74%, peak 4 (apo AI−1, pI 5.42) for about 13%, and peak 5 (apo AI−2, pI 5.37) for about 5%. In some subjects, the proportion of proapo AI was increased. Variations of the ratio of the different apo AI isoforms may be due to modification of the fractional catabolic rate of this apolipoprotein. The procedure proposed in this study may be useful for the evaluation of quantitative abnormalities in apo AI isoforms involved in the development of coronary heart diseases (CHD).
Capillary electrophoresis of herbicides: IV. Evaluation of octylmaltopyranoside chiral surfactant in the enantiomeric separation of fluorescently labeled phenoxy acid herbicides and their laser‐induced fluorescence detection
Authors: Mechref Yehia , El Rassi Ziad 
Published: May 2005

AbstractA novel chiral nonionic surfactant, namely octyl‐b‐D‐maltopyranoside (OM), was evaluated in chiral capillary electrophoresis of fluorescently labeled phenoxy acid herbicides. The labeling of the analytes with 7‐aminonaphthalene‐1,3‐disulfonic acid (ANDSA) permitted a concentration detection limit of 5 × 10−10 M using laser‐induced fluorescence detection. This limit of detection allowed the determination of ultradiluted solutions of the ANDSA‐derivatized phenoxy acid herbicides whose concentration was as low as 10−11 M (i.e. 2.2 ppt) by applying the concept of field‐amplified sample stacking (FASS). The sample injection by FASS did not adversely affect separation efficiencies, resolution and reproducibility of the electroporetic system. The tagging of the phenoxy acid herbicides with ANDSA increased the hydrophobicity of the analytes, thus favoring an enhanced solubilization of the derivatized herbicides in the OM micellar phase. The net results of this effect were a much shorter analysis time and an improved enantiomeric resolution of the derivatives when compared to underivatized phenoxy acid herbicides. The optimum surfactant concentration required for maximum resolution decreased with increasing hydrophobicity of the analyte, with the least hydrophobic analyte requiring higher surfactant concentration. Because of the two permanently charged sulfonic acid groups of the ANDSA tag, the pH of the running electrolyte had little effect on the enantiomeric resolution of the derivatized herbicides. Due to its salting‐out effect and increasing the micellized surfactant concentration, increasing the ionic strength of the running electrolyte increased the enantiomeric resolution of the least hydrophobic analytes. Conversely, increasing the percent methanol in the running electrolyte decreased the enantiomeric resolution of the least hydrophobic analytes due to decrease strength of solute‐micelle association. For hydrophobic analytes, existed an optimum percent of methanol existed for maximum enantiomeric resolution.
Separation of fungicides by micellar electrokinetic capillary chromatography
Authors: Penmetsa Kumar V. , Leidy Ross B. , Shea Damian 
Published: May 2005

AbstractCapillary electrophoresis with ultraviolet detection (CE/UV) of selected fungicides (carbendazim, metalaxyl, propiconazole, and vinclozolin) using different buffer compositions was investigated. Capillary zone electrophoresis (CZE) with 10 mM sodium phosphate (pH 7.0) was not useful in separating the four fungicides used in this study. However, the four fungicides were well resolved by employing micellar electrokinetic capillary chromatography (MEKC). Among the two surfactants tested in MEKC, bile salts provided better separation compared to sodium dodecyl sulfate (SDS). A buffer consisting of 10 mM sodium phosphate with 100 mM sodium cholate and 10% methanol (pH 7.0) gave best results; excellent separation of the four compounds was achieved in less than 15 min. The CE/UV method was validated by analyzing deionized and lake‐water samples fortified with known concentrations of the four fungicides. Average recoveries of the fungicides in lake water at 4 μg/L level ranged from 42 to 87%.