bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of RNA by capillary electrophoresis
Authors: Skeidsvoll Jarle , Ueland Per Magne
Published: May 2005

Analytical 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

The 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

We 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

The 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

Fifty‐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 Jonesia

Presented 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

A 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

Aracytidine (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

In 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

Resolution 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

The 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

Quantitative 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

A 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

Capillary 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%.
The effect of cyclodextrin modifiers on electrophoretic separation of aromatic hydrocarbons
Authors: Nguyen An‐Lac , Luong John H. T.
Published: May 2005

Cyclodextrin‐modified capillary electrophoresis has been developed for separation and analysis of benzene and its derivatives. The procedure used a mixture of negatively charged sulfobutyl ether‐β‐cyclodextrin (SBβCD) and neutral hydroxypropyl‐β‐cyclodextrin (HPβCD) to effect differential distribution (partitioning) of the aromatic hydrocarbons between the buffer and CD phases. In 80 mM phosphate buffer, containing 15 mM SBβCD and 5 mM HPβCD, benzene, toluene, ethyl benzene and three xylene isomers (BTEXs) were well resolved with a number of theoretical plates well above 100 000, for 50 cm of effective length. Some halogenated benzenes were also observed to separate well from the BTEX components to indicate their suitability as an internal standard for BTEX analyses. Equilibrium complexation models were used for investigating the effect of the cyclodextrin(s) at different concentrations and temperature on the electrophoretic mobility.
Analysis of brevetoxins by micellar electrokinetic capillary chromatography and laser‐induced fluorescence detection
Authors: Shea Damian
Published: May 2005

Micellar electrokinetic capillary chromatography (MEKC) with laser‐induced fluorescence (LIF) detection was used to measure four red tide brevetoxins at sub‐attomole levels. The separation of four brevetoxins by MEKC was achieved with a sodium borate/sodium dodecyl sulfate buffer at pH 9.3. Brevetoxins with a terminal alcohol group were derivatized with an acyl azide coumarin to form stable, highly fluorescent products. Brevetoxins with a terminal aldehyde group were reduced to the alcohol with sodium borohydride prior to derivatization with the coumarin. Three derivatized brevetoxins (PbTx‐3, PbTx‐5, and Pb Tx‐9) were separated by MEKC and detected using He/Cd laser excitation at 354 nm and fluorescence emission at 410 nm. A fourth brevetoxin (Pb Tx‐2) was converted to Pb Tx‐3 prior to derivatization and was then determined by subtraction. Instrumental detection limits for all four toxins were approximately 0.10 fg or about 106‐fold more sensitive than existing liquid chromatographic methods. Brevetoxins were isolated from cell cultures and fish tissue using an alumina column/gel‐permeation chromatography procedure. Method detection limits for the brevetoxins in fish tissue were approximately 4 pg/g. These method detection limits are at least 100‐fold better than previous chromatographic and/or electrophoretic methods. The MEKC‐LIF method reported here allows measurement of brevetoxins at the trace levels considered critical for understanding toxin metabolism and mode of action.
Analysis of toxic drugs by capillary electrophoresis using polyacrylamide‐coated columns
Authors: Jinno Kiyokatsu , Han Yinghong , Sawada Hirokazu
Published: May 2005

Toxic drugs, including barbiturates and benzodiazepines, were analyzed using polyacrylamide‐coated columns in capillary electrophoresis (CE). The separations were carried out in absence of electroosmotic flow. Seven kinds of barbiturates were successfully separated with the coated columns in free solution without further additives. Benzodiazepines, the electrically neutral solutes, were introduced onto the coated column, and separated in presence of sodium dodecyl sulfate above its critical micelle concentration in the running buffer. This CE method offered fast and efficient separation of more hydrophobic solutes, such as benzodiazepines. The separation of seven barbiturates was studied in linear (noncross‐linked) polyacrylamide solutions and in acrylamide / N‐isopropylacrylamide (AA/IPAA) copolymer solutions to explore the effect of isopropyl groups in the AA/IPAA copolymer chain.
Improvement of the solubilization of proteins in two‐dimensional electrophoresis with immobilized pH gradients
Authors: Rabilloud Thierry , Adessi Céline , Giraudel Anne , Lunardi Joël
Published: May 2005

Membrane and nuclear proteins of poor solubility have been separated by high resolution two‐dimensional (2‐D) gel electrophoresis. Isoelectric focusing with immobilized pH gradients leads to severe quantitative losses of proteins in the resulting 2‐D map, although the resolution is usually high. Protein solubility could be improved by using denaturing solutions containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and detergents (both nonionic and zwitterionic). The usefulness of thiourea‐containing denaturing mixtures is shown for microsomal and nuclear proteins as well as for tubulin, a protein highly prone to aggregation.
Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping
Authors: Loo Rachel R. Ogorzalek , Mitchell Charles , Stevenson Tracy I. , Martin Stephen A. , Hines Wade M. , Juhasz Peter , Patterson Dale H. , Peltier John M. , Loo Joseph A. , Andrews Philip C.
Published: May 2005

Matrix‐assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin‐layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of α‐ and β‐chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one‐dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time‐lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa α‐subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)‐polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.
A comparison of selected mRNA and protein abundances in human liver
Authors: Anderson Leigh , Seilhamer Jeff
Published: May 2005

In order to obtain an estimate of the overall level of correlation between mRNA and protein abundances for a well‐characterized pharmaceutically relevant biological system, we have analyzed human liver by quantitative two‐dimensional electrophoresis (for protein abundances) and by Transcript Image methodology (for mRNA abundances). Incyte's LifeSeq™ database was searched for expressed sequence tag (EST) sequences corresponding to a series of 23 proteins identified on 2‐D maps in the Large Scale Biology (LSB) Molecular Anatomy™ database, resulting in estimated abundances for 19 messages (4 were undetected) among 7926 liver clones sequenced. A correlation coefficient of 0.48 was obtained between the mRNA and protein abundances determined by the two approaches, suggesting that post‐transcriptional regulation of gene expression is a frequent phenomenon in higher organisms. A comparison with published data (Kawamoto, S., et al., Gene 1996, 174, 151–158) on the abundances of liver mRNAs for plasma proteins (secreted by the liver) suggests that higher abundance messages are strongly enriched in secreted sequences. Our data confirms this: of the 50 most abundant liver mRNAs, 29 coded for secreted proteins, while none of the 50 most abundant proteins appeared to be secreted products (although four plasma and red blood cell proteins were presents in this group as contaminants from tissue blood).
Characterisation of wool intermediate filament proteins separated by micropreparative two‐dimensional electrophoresis
Authors: Herbert Ben R. , Molloy Mark P. , Yan Jun X. , Gooley Andrew A. , Bryson Warren G. , Williams Keith L.
Published: May 2005

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post‐translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two‐dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two‐dimensional electrophoresis of alkaline phosphatase‐treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post‐separation analysis, micropreparative two‐dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4–7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)‐separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2–4.9, 4.5–5.4, 5.0–6.0 and 5.6–6.6.
Protein expression changes associated with radiation‐induced neoplastic progression of human prostate epithelial cells
Authors: Prasad Sarada C. , Thraves Peter J. , Dritschilo Anatoly , Kuettel Michael R.
Published: May 2005

Carcinogenic progression in most epithelial systems is a multistep process and presents as numerous (un)stable intermediate stages prior to the development of a fully malignant phenotype. Recently, we reported the neoplastic transformation of an SV40 immortalized, neonatal human prostate epithelial cell line (267B1) by multiple exposures to X‐rays [1, 2]. The parental 267B1 cells acquired anchorage‐independence and exhibited morphological transformation following exposure to two consecutive doses of 2 Gy. Exposure of either the parental 267B1 cells or the anchorage‐independent derivatives (F3‐SAC) to a total dose of 30 Gy of X‐rays yielded tumorigenic transformants (267B1‐XR and 267B1‐SXR, respectively). All of these radiation‐treated derivatives (F3‐SAC, 267B1‐XR, and 267B1‐SXR) were characterized by reduced cell size and poorly organized actin stress fibers [2, 3]. The present study examines the protein expression changes associated with cytoskeletal alterations during the different steps of neoplastic progression induced by X‐rays in the in vitro human prostate cell system. This analysis was achieved by using the high resolving power of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in the 267B1, F3‐SAC, 267B1‐XR, and 267B1‐SXR cells. We report changes in the expression of gelsolin in the partially transformed, anchorage‐independent, nontumorigenic (F3‐SAC) cells and a progressive loss of expression of tropomyosin isoforms (TM‐1 and TM‐3), and myosin light chain‐2 (MLC‐2) in the tumorigenic (267B1‐XR; 267B1‐SXR) cells, respectively. In contrast, our results demonstrate that the levels of the small GTP‐binding protein Rho‐A, an active participant in the actin stress fiber organization, are not altered during neoplastic progression of these 267B1 cells. Thus the changes in synthesis of gelsolin, tropomyosins, and MLC‐2 provide a rationale for the alterations in the actin stress fiber formation and reduction in cell size during the exposure of prostate epithelial cells to multiple doses of X‐rays.
Capillary electrophoretic separation of tricyclic antidepressants using charged carboxymethyl‐β‐cyclodextrin as a buffer additive
Authors: Spencer Brian J. , Zhang Wenbin , Purdy William C.
Published: May 2005

Charged carboxymethyl‐β‐cyclodextrin was successful in the capillary electrophoretic separation of a series of tricyclic antidepressants. The cyclodextrin alone was successful in the separation of carbamazepine, protriptyline, desipramine, clomipramine, and opipramol using a 3‐(trimethoxysilyl)propyl methacrylate capillary coating to reduce the electroosmotic flow. The ideal buffer pH was found to be in the range of 6–7 and the ideal cyclodextrin concentration to be 10 mM. All nine antidepressants were resolved using the charged cyclodextrin in the micellar electrokinetic chromatography (MEKC) mode with sodium dodecyl sulfate as the surfactant. Neither the cyclodextrin nor the surfactant alone were successful in resolving the whole series of compounds under investigation but a combination of both produced the separation. Separations were performed on a linear polyacrylamide coated capillary. The ideal pH of the buffer was in the range of 5–7.
Isolation of subcellular‐sized particles separated by electrophoresis in dilute polymer solution, using commercial electrophoresis apparatus with intermittent scanning of fluorescence
Authors: Chen Nong , Chrambach Andreas
Published: May 2005

Resolution of subcellular‐sized particles in electrophoresis employing semidilute polymer solutions as “sieving media” improves as the polymer concentration is decreased. Therefore, the previously reported conditions of preparative electrophoresis of microsomes, using concentrated (12%) polyvinylpyrrolidone (PVP) solutions, while solving the problem of non‐entrance of large particles into “sieving media”, do not provide adequate resolving capacity, as exemplified by failure of the microsome preparation used, to resolve in the manner of gels or dilute solutions. The present report provides the conditions under which the HPGE‐1000 apparatus can be preparatively applied when the electrophoretic separation is effectively conducted in a dilute polymer solution. The isolation of three microsome components under those conditions constitutes the first application of “particle sieving”, i.e., a separation due preponderantly to size and shape differences, at a preparative scale.
Alternative method of subtyping the A subunit of coagulation factor XIII (FXIIIA)
Authors: Kido Akira , Oya Masakazu , Jin Zeming
Published: May 2005

An alternative isoelectric focusing method for the A subunit of coagulation factor XIII (FXIIIA) subtyping is described which employs three different carrier ampholytes pH 4–6.5, pH 5–6 and pH 5–8 (2:2:1), and sample application at the anode. With this technique ten common subtypes, including a variant, were clearly and reliably identified in Japanese and Chinese population samples.
Dilated cardiomyopathy‐associated proteins and their presentation in a WWW‐accessible two‐dimensional gel protein database
Authors: Plei Klaus‐Peter , Söding Peter , Sander Stefan , Oswald Helmut , Neuß Michael , Regitz‐Zagrosek Vera , Fleck Eckart
Published: May 2005

High resolution two‐dimensional electrophoresis (2‐DE) and computer‐assisted image analysis were used to screen 13 patients suffering from dilated cardiomyopathy (DCM) versus 15 control patients for quantitative and qualitative differences in their myocardial protein expression. Right atrial tissue samples were obtained from end‐stage failing explanted hearts and control hearts. Fifty‐two spots differed significantly in average intensity between the DCM and the control groups. Myosin light chain 2, ventricular (MLC2) and heat shock protein HSP 27 were identified by protein microsequencing and gel map comparison with other databases. These proteins were found to be characteristic protein markers for DCM in the right atrium. In DCM patients, the spot intensity (protein abundance) of MLC2 is increased to 336% and HSP 27 is decreased to 59%, compared to the control group. The HEART‐2DPAGE, a World Wide Web‐accessible 2‐DE database, was used and extended for the presentation of these disease‐associated proteins. Retrievable via Internet we present a list of disease‐associated proteins, their altered level of expression in DCM, and their position on a right atrial protein pattern. The accession number to protein sequence databases confers a connection to databases like SWISS‐PROT to obtain a detailed functional and structural description of disease‐associated proteins. New DCM‐associated proteins are detected and their presentation in a 2‐DE gel protein database is described.
High resolution two‐dimensional electrophoretic analysis of urinary proteins of patients with prostatic cancer
Authors: Grover Phulwinder K. , Resnick Martin I.
Published: May 2005

In an attempt to identify marker(s) for prostatic cancer, proteins in urines of normal and prostatectomized males and in men with cancerous prostate were analyzed. Only urines collected with protease inhibitors were examined. Two‐dimensional (2‐D) gel electrophoresis was used for high resolution separation of proteins and the electrophoretograms were either developed by double stain or the proteins were electrophoretically transferred onto nitrocellulose for immunological identification. The pool of each group exhibited similar relative positions of major protein spots. The study of normal and prostatectomized men identified two proteins denoted as A (36 kDa, pI 6–6.5) and B (23 kDa, pI 6.6), which were undetectable in the latter group. A visual comparison of the patterns of normals and patients with cancerous prostate revealed that both these proteins were undetectable in urines of men with malignant prostatic carcinoma (PCA) and benign prostatic hyperplasia (BPH) and hence may be useful in identifying prostatic carcinoma. Also, while protein C 43.5 kDA, pI 6–6.6) was discerned in normals its abundance, along with those of proteins D (40 kDa, pI 6–6.4) and E (26.5 kDa, pI 6–6.7), appeared to be higher in BPA than in PCA. Protein F (18–28 kDa, pI 4–5.5) was found in patients with BPH but was undetected in normals and men with PCA. Hence, it may become useful in distinguishing BPH from PCA. All the proteins, A to F, appear to be previously unidentified. Their further characterization is warranted.
Analysis of proteins from different phase variants of the entomopathogenic bacteria Photorhabdus luminescens by two‐dimensional zymography
Authors: Ong Kevin L. , Chang Frank N.
Published: May 2005

Two‐dimensional zymography which combines two‐dimensional electrophoresis with zymography was used to analyze proteases and other proteins produced by different phase variants of two strains of Photorhabdus luminescens. Both the primary and secondary phases of P. luminescens strains Hp and Hm secreted proteases. The protease in P. luminescens Hp has a molecular weight (Mr) of 57 000 and an isoelectric point (pI) of 4.4 whereas that in P. luminescens Hm has an Mr of 59 000 and pI of 4.9. Several putative protease degradation products were clearly visible in the zymograms from both bacterial strains. Two‐dimensional zymography also showed that several secretory proteins were present only in particular phase variants and therefore could be used as specific markers. Unexpectedly, the two‐dimensional zymography revealed that a nonsecretory protease with an Mr of 47 000 and a pI of 4.0 was present in the cell extracts of all phases of both P. luminescens Hp and Hm. The application of the two‐dimensional zymography for the identification of other enzymes was also discussed.
Use of a zwitterionic cyclodextrin as a chiral agent for the separation of enantiomers by capillary electrophoresis
Authors: Lelièvre Frédéric , Gueit Carole , Gareil Pierre , Bahaddi Youssef , Galons Hervé
Published: May 2005

The purity and enantioselectivity of a novel chiral agent, the zwitterionic mono‐(6‐δ‐glutamylamino‐6‐deoxy)‐β‐cyclodextrin (β‐CD‐Glu), were studied by capillary electrophoresis. Chiral separation of the enantiomers of chlorthalidone was obtained at pH 2.3, a pH at which β‐CD‐Glu is partially protonated. Comparison with the cationic mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin (β‐CD‐NH2) enantioselectivity clearly shows that the greater the difference in mobility between the free analyte and the analyte‐cyclodextrin complex, the better the resolution. Hydrobenzoin enantiomers were separated at pH 11.2, a pH at which β‐CD‐Glu is anionic. Under these conditions, the migration order was opposite to that observed in the presence of β‐CD‐NH2 at pH 2.3. When no separation was obtained directly with β‐CD‐Glu, a dual cyclodextrin system was developed. Carprofen enantiomers were resolved at pH 2.3 in the presence of a β‐CD‐Glu/trimethyl‐β‐cyclodextrin (TM‐β‐CD) system in which the charged CD confers a non‐zero mobility to the analyte, while the neutral CD allows chiral recognition.
Chiral separation of DL‐peptides and enantioselective interactions between teicoplanin and D‐peptides in capillary electrophoresis
Authors: Wan Hong , Blomberg Lars G.
Published: May 2005

Teicoplanin has been evaluated as a selector for enantioseparation of di‐ and tripeptide derivatives in capillary electrophoresis. Separation variables such as type of buffer, pH, concentrations of teicoplanin and organic modifier were examined. Optimal separation conditions were obtained by means of factorial design experiments. The effects of teicoplanin concentrations below and above its critical micellar concentration (CMC) and of acetonitrile (ACN) on the separation were demonstrated. The use of a high concentration of ACN resulted not only in increased selectivity, but also in improved separation efficiency. Electroosmotic flow was observed to be largely independent of the concentrations of teicoplanin under the optimized conditions. Good repeatability of migration times was obtained. The interactions between teicoplanin and D and L peptides were studied, and it was found that, for some peptides, teicoplanin exhibited enantioselective interaction only with the D‐form. Somewhat lower separation efficiencies were thus observed for the strongest interacting (D‐form) peptides. Chiral separation of 15 DL‐peptide derivatives was achieved in less than 10 min.
A quantitative relationship between capacity factor and selector concentration in capillary electrophoresis and high performance liquid chromatography: Evidence from the enantioselective resolution of benzoin using human serum albumin as a chiral selector
Authors: Lloyd David K. , Ahmed Abdelwab , Pastoré Florent
Published: May 2005

Many selectors are used both in pressure‐driven liquid chromatography (LC) and in electrokinetic chromatography (EKC), particularly chiral species such as cyclodextrins and proteins. It should be possible to readily apply information gleaned using one technique to the other, since in both techniques the underlying molecular interactions which lead to separations are expected to be the same. Superficially this may be the case, but an exact transfer of operating conditions, i.e., background electrolyte (BGE) composition/mobile phase composition, assuming that these meet certain minimum requirements for each technique, is not often possible. To investigate the reason for this we have measured retention (K′) of a neutral solute (racemic benzoin) in HPLC and EKC using an identical range of BGE/mobile phase conditions in both techniques. The selector used was human serum albumin. The K′ measurements obtained for each benzoin enantiomer were consistently higher in HPLC than in EKC. This can be explained very simply if one considers that retention in both systems is related to the selector concentration [S], by the expression K′=K[S], where K is the affinity constant. In EKC, [S] is simply the concentration of free selector in the BGE, while in LC, [S] = mp/Vo, where mp is the number of moles of accessible selector, and Vo is the column void volume. In LC, [S] is generally considerably higher than in EKC, leading to larger values of K′.
Enantiomer separation of disopyramide with capillary electrophoresis using various cyclodextrins
Authors: Juvancz Zoltán , Markides Karin E. , Jicsinszky László
Published: May 2005

Enantiomers of disopyramide display different biological actions, and therefore chiral selective analysis is necessary. Fifteen different cyclodextrins (CDs) and CD derivatives were tested as capillary electrophoresis (CE) additives for the chiral separation of disopyramide. Eleven types of CDs showed chiral recognition features and four types had a baseline or close to baseline separation. The best resolution (Rs = 3.0) was with 15 mM carboxymethylated β‐CD (pH 4.9). A sharp decrease in the selectivity of γ‐phosphate (γ‐PhoCD) was observed in the pH range of 2–3, indicating a structural change of γ‐PhoCD. The enantiomers of disopyramide were separated in its ionized as well as neutral forms using acidic substituted CDs. The results show that the size of the CD cavity can not be used as a guide to estimate chiral separations, suggesting a more complex separation mechanism of these CDs towars disopyramide.
Enantioselective determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in serum, urine and hair by capillary electrophoresis
Authors: Frost Michael , Köhler Helga , Blaschke Gottfried
Published: May 2005

A capillary electrophoresis method has been developed for the enantioselective determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in serum and urine. After addition of the internal standard diphenhydramine, the serum and urine samples were extracted at pH 9–10 withn‐hexane. Different cyclodextrins were tested for use as chiral selectors. The best results for simultaneous separation of methadone and EDDP were obtained using heptakis‐(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DIMEB). Data on the accuracy and precision of the method are presented. The method was applied to serum, urine, and hair samples, from individuals undergoing methadone therapy, under consideration of multi‐drug abuse.
Separation of tumor necrosis factor α isoforms by two‐dimensional polyacrylamide gel electrophoresis
Authors: Watts Alan D. , Hunt Nicholas H. , Hambly Brett D. , Chaudhri Geeta
Published: May 2005

The mouse macrophage cell‐line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose‐dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two‐dimensional (2‐D) electrophoresis with immobilised pH gradient 3–10 linear gels as the first dimension. After radiolabelling of cells with 35S, both cell‐associated and supernate‐associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2‐D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.
Polymorphism of 5′ of the catalase gene in Hungarian acatalasemia and hypocatalasemia
Authors: Góth László , Vitai Márta
Published: May 2005

The amplified fragment length polymorphism of Hinfl on the promoter region of the catalase gene in Hungarian acatalasemic and hypocatalasemic patients yielded three different patterns with five bands in total. The sequence analyses revealed A‐to‐T, C‐to‐A, and C‐to‐T mutations at positions −21, −20, and −18 upstream of the translational initiation site. The −21 A‐to‐T mutations were more frequent in acatalasemic and hypocatalasemic patients (36/2) than in controls (18/14). This mutation had been detected in Japanese acatalasemic patients while the other two are novel mutations. Two extra bands in the Hinfl pattern are due to star‐like activity that cleaved a G/ATTT sequence at position −4 to 0 upstream of the initiation site.
Cellular electrophoretic mobility data: A first approach to a database
Authors: Slivinsky George G. , Hymer Wesley C. , Bauer Johann , Morrison Dennis R.
Published: May 2005

Cellular electrophoretic mobility values of 288 types of eucaryotic cells were collected from literature published worldwide by a series of authors during the past forty years and arranged in a list. This list contains well‐known recent electrophoretic results and also data that cannot be found anymore with modern literature retrieval systems. It will be a valuable help for scientists trying to purify cell populations. In addition, it confirms the observation that most eucaryotic cells have very similar electrophoretic mobilities, ranging from 40% above to 50% below the electrophoretic mobility of human erythrocytes, and thus reinforces the suggestion that electrophoretic mobilities of eucaryotic cells are subjected to strong biological controls.