bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of bioproducts by free‐flow zone electrophoresis: Choice of processing parameters
Authors: Roux‐De Balmann Hélène , Cerro Rose‐Marie , Sanchez Victor
Published: May 2005

Free‐flow zone electrophoresis may be used to purify biological samples, due to differences in electrophoretic mobility, in absence of a matrix – most frequently a gel – thus enabling the biological integrity of even fragile molecules to be preserved. However, the process is more complicated than its principle suggests due to different transport phenomena interfering with electrophoretic migration, with the resultant separation depending both on separation effects and dispersive phenomena. The physical origin of the main effects involved was identified. Mathematical expressions were proposed to estimate the influence of the crescent effect and electrohydrodynamics on the process. In this paper, these equations are used to determine the minimum difference in electrophoretic mobility required for a separation to be achieved with respect to the processing parameters. A methodology is proposed which defines the conditions under which the difference in electrophoretic mobilities equals that calculated when considering the influence of dispersive phenomena. Optimized separations of the whey proteins lactoferrin and albumin, known to interact strongly, and the purification of a monoclonal antibody from a mouse ascitic fluid illustrate the approach.
Preparation of Golgi subfractions with free‐solution isotachophoresis: Analysis of sphingomyelin synthesis in Golgi subfractions from rat liver
Authors: Hofmeister Robert , Böttcher Alfred , Schmitz Gerd
Published: May 2005

A new displacement electrophoresis technique, termed free‐solution isotachophoresis (FS‐ITP) was used for the analysis of sphingolipid metabolism in Golgi subfractions. The discontinuous electrolyte system enables tissue‐derived membrane vesicles to be separated and purified due to their polarity patterns in a mobility gradient. In this study total Golgi apparatus obtained from rat liver by discontinuous density gradient centrifugation was subfractionated by preparative FS‐ITP, yielding enzymatically active cis‐, medial‐, and trans‐Golgi subfractions. These membrane vesicles were assayed by the following established enzyme marker activities: NADH cytochrome c reductase (cis‐Golgi), NADP phosphatase (medial‐Golgi), and thiamine pyrophosphatase (trans‐Golgi). The activity of phosphatidylcholine:ceramide phosphocholine transferase, a sphingomyelin synthesizing enzyme, is attributed to the cis‐ and medial‐Golgi‐derived subfractions. Analysis of Golgi lipids revealed a decline in membranous ceramide along the cis‐ to trans‐Golgi polarity axis. Furthermore, significant amounts of newly synthesized sphingomyelin and diacylglycerol are transferred from the medial/cis‐ to the trans‐Golgi compartment. The FS‐ITP system is well suited for micropreparative experimental applications, as demonstrated by studies on phosphatidylcholin:exeramide phosphocholine transferase activity in Golgi membrane vesicles of rat liver obtained by FS‐ITP.
Separation and microgram‐scale isolation of sea urchin egg granules by electrophoresis in polyvinylpyrrolidone solution, using horizontal gel electrophoresis apparatus with fluorescence detector
Authors: Chen Nong , Radko Sergey P. , Chrambach Andreas
Published: May 2005

A homogenate of sea urchin (Lytechinus pictus) eggs rich in exocytotic membrane vesicles (granules) was subjected to analytical and preparative electrophoresis in the commercial automated horizontal gel electrophoresis apparatus (HPGE‐1000, LabIntelligence, Belmont, CA) capable of intermittent scanning of the migration path, using buffered solutions of polyvinylpyrrolidone (PVP). The nonfluorescent granules were detected by “fluorescence reduction”, i.e., a decrease of fluorescence intensity due to the absorbance and/or light scattering properties of the particle. Granules migrated at linear migration rates in buffers ranging from 0 to 2.5% PVP. Two bands were observed and optimally separated in 1.5% PVP solution. As shown by sodium dodecyl sulfate (SDS)‐polypeptide patterns, the material recovered from the bands was qualitatively indistinguishable from the two major fractions A and C of granules previously separated by free‐flow electrophoresis in the absence of polymer. Ferguson plot analysis failed to provide the sizes of the granules in view of the narrow PVP concentration range available for mobility measurement and the unavailability of chemically homogeneous size standards.
Surface modification of superparamagnetic nanoparticles (Ferrofluid) studied with particle electrophoresis: Application to the specific targeting of cells
Authors: Sestier Claude , Da‐Silva Maria Fatima , Sabolovic Domagoj , Roger Jackie , Pons Jean Noël
Published: May 2005

Colloidal aqueous suspension of superparamagnetic nanoparticles (9 nm in diameter) composed of maghemite (γ Fe2O3) and forming an ionic ferrofluid in aqueous solution are covalently coupled with lectins, enzymes or antibodies, using specific thiol chemistry. The surface charge modifications of nanoparticles, caused by ligand coupling, were monitored by measuring their electrophoretic mobilities using laser‐Doppler velocimetry. Particle electrophoretic mobility (PEM) changes are shown to correlate well with the amount of ligand fixed on the particles, as probed by its biological activity. The PEM method provides a useful tool to optimize ligand immobilization at the surface of nanoparticles, and may be advantageous when biological activity measurements are not convenient.
Cell electrophoretic mobility and glycerol lysis of human erythrocytes in various diseases
Authors: Hashimoto Nobuya , Fujita Shuzo , Yokoyama Toru , Ozawa Yohinori , Kingetsu Isamu , Kurosaka Daitaro , Sabolovic Domagoje , Schuett Wolfgang
Published: May 2005

Using an automated cell electrophoresis system equipped with an image processor, we studied electrophoretic mobilites of erythrocytes of healthy donors and of patients suffering from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), hypergammaglobulinemia and diabetes mellitus (DM). On average, erythrocytes from SLE patients showed mean electrophoretic mobilities (EPM) which were significantly lower (p < 0.005) than the EPM of red blood cells of normal donors. Evaluation of mean EPM and standard deviations revealed that three groups of SLE patients could be distinguished regarding the electrophoretic behavior of their erythrocytes. Some patients had red blood cells with normal EPM, others had erythrocytes with significantly reduced EPM, and a third group appeared to have both kinds of erythrocytes. In addition, erythrocytes of various SLE patients showed enhanced resistance to lysis by glycerol and their membranes contained less quantities of band 3 proteins.
Experimental study on temperature distribution within a wide‐gap continuous free‐flow electrophoresis chamber
Authors: Wang Haibo , Zhang Zhiyuan , Jiang Yuanda , Wu Hanji
Published: May 2005

The temperature distribution within a wide‐gap (3 mm) continuous free‐flow electrophoresis (CFFE) chamber was investigated by direct measurements. The walls of the chambers consisted of Plexiglas and thermocouples were used as temperature sensors. The study of different operating and heat dissipating conditions revealed that lateral temperature gradients arise in CFFE chambers in addition to an axial increase in temperature of 0.25°C per Watt of electric power and per minute of residence time in the absence of cooling.
Towards predicting mobility and resolution in polymeric media: Some first steps
Authors: Chrambach Andreas , Radko Sergey P.
Published: May 2005

Particle size‐dependent retardation (“molecular sieving”) in electrophoresis can be achieved in polymer solutions or gels. In the semidilute concentration range of polymer solutions, mobility of “rigid, spherical” particles can be predicted from their size (in the size range less than 20 nm radius), the screening length specific for the particular polymer and a constant that can be experimentally determined for a polymer. That constant could be universal for all hydrophilic uncharged polymers. Band spreading in polymer solutions is constant over a range of particle sizes and polymer concentrations and increases beyond that range. Presumably, the critical division between the two ranges occurs when the diameter of the particle exceeds the screening length of the polymer network. Resolution, defined as separation divided by the sum of bandwidths, can thus be predicted for a limited particle size and polymer concentration range. In gels, resolution can be estimated from the slopes and free mobility intercepts of the Ferguson plot. However, the previous computation of resolution needs to be revised in view of the fact that band spreading in gels proceeds in proportion to time, presumably through interaction with the polymer, and not as a function of the square root of time as would be the case if band spreading resulted from diffusion.
Creutzfeldt‐Jakob disease: Clinical features, epidemiology and tests
Authors: Knight Richard
Published: May 2005

Creutzfeldt‐Jakob disease (CJD) is now recognized to occur in four main forms: classical sporadic, genetic, iatrogenic and new variant. These are briefly described. The evidence that new variant disease is due to bovine spongiform encephalopathy (BSE) is reviewed. In CJD, disease and infectivity are associated with the accumulation of an abnormal form of a normal cellular protein. Protein analysis and the study of the gene which encodes for this protein have therefore played an important role in understanding CJD. Diagnostic tests for CJD are discussed with particular reference to the detection of the 14‐3‐3 protein in the cerebrospinal fluid (CSF).
Correlation of plasma protein separation patterns obtained by two‐dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis
Authors: Manabe Takashi , Miyamoto Hiromitsu , Oota Hideto , Toyota Yosuke
Published: May 2005

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)‐filled capillary electrophoresis with SDS (SDS‐CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2‐D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.
Membrane‐mediated sample loading for automated DNA sequencing
Authors: Cassel Sallie M. , Guttman András
Published: May 2005

A new and improved sample loading method for DNA sequencing gels using oligo/polynucleotide binding membranes is described. The labeled DNA sequencing fragments were spotted onto a membrane sample loader, which was then placed in contact with the precast polyacrylamide separation gel. In this way, the time‐consuming sample well loading by the tedious pipetting procedure was avoided. The spotted DNA fragments remain immobilized on the membrane until the separation process is initiated by the application of the electric field (an active DNA release mechanism). This novel technique enables sample loading outside of the separation platform, thereby allowing full utilization of various automated sample preparation and liquid handling (robotics) systems, resulting in “real” automated DNA sequencing. The loaded membranes can be stored for more than 24 hours for later use.
Nonradioactive Northern blotting with biotinylated and digoxigenin‐labeled RNA probes
Authors: Meltzer Jon C. , Sanders Veronica , Grimm Paul C. , Chiasson Nathalie , Hoeltke Hans‐Joachim , Garrett Karryn L. , Greenberg Arnold H. , Nance Dwight M.
Published: May 2005

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn‐around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)‐labeled RNA probes from nylon membranes. One protocol utilizes a phosphate‐buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip‐EZ™. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin‐labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called ChemLink™, that quickly and conveniently labels RNA for use in Northern blotting.
A 33 bp minisatellite repeat upstream of the ‘mutated in colon cancer’ gene at chromosome 5q21
Authors: Bugert Peter , Kenck Christiane , Kovacs Gyula
Published: May 2005

We have found a 33 bp minisatellite repeat in the 5′‐flanking region of the mutated in colon cancer (MCC) gene at chromosome 5q21. Southern blot experiments demonstrated the locus specificity of the repeat. The number of repeat units varied between 5 and 11 with a heterozygosity of 0.56. The sequence 5′‐AGG AGT GTG AAT GGG GCA TAG TGA ATG AGG GGA‐3′ of the repeat units does not match the consensus sequence of chi‐related minisatellites. The minisatellite is not expressed as part of a gene transcription unit. However, it can be used as a tool for the detection of allelic changes at chromosome 5q21 on standard agarose gels.
Ultrafast DNA analysis by capillary electrophoresis/laser‐induced fluorescence detection
Authors: Müller Odilo , Minarik Marek , Foret Frantisek
Published: May 2005

The limits of ultrafast DNA analysis by CE were determined by investigating the influence of the effective capillary length and the electric field strength on the analysis time for a given peak resolution (10 bp). In accordance with theory, the use of a fast ramp power supply for narrow plug electrokinetic injection was found to be essential to minimize the extra column effects on peak dispersion. Two major column dispersion factors, logitudinal diffusion and thermal dispersion, were determined experimentally, as well as the influence of the electric field strength on the electrophoretic mobilities and diffusion coefficients of DNA. It was found that higher field strengths can be applied with lower thermal dispersion than predicted by classical CE models. This was attributed to the faster mass transport in the radial direction due to field‐induced DNA orientation. Short capillaries (∼ 3–7 cm effective length) and moderate to high electric field strengths (∼ 600–800 V/cm) were used to perform a series of fast DNA separations. The dsDNA fragment standards ϕX174/HaeIII and pBR322/HaeIII were separated within 30 s. The possibility for fast mutation detection was demonstrated using constant denaturant capillary electrophoresis (CDCE) for the analysis of a single base mutation in mitochondrial DNA in 72 s. The potential for fast DNA sequencing was illustrated by separating 300 ssDNA fragments within 180 s.
The combined effect of acetonitrile and urea on the separation of polycyclic aromatic hydrocarbons using sodium dioctyl sulfosuccinate in electrokinetic chromatography
Authors: Luong John H. T.
Published: May 2005

Sodium dioctyl sulfosuccinate (DOSS) or sodium di2‐ethylhexyl sulfosuccinate, a relatively nontoxic and negatively charged surfactant, was selected and optimized for the capillary electrophoretic separation of the 16 Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs). This pseudostationary phase displayed selectivities for PAHs which were somewhat similar to the C18 phase in reversed‐phase high performance liquid chromatography (HPLC). At high DOSS concentrations (> 30 mM), the hydrophobic interaction between DOSS and PAHs was pronounced and led to stronger retention of the latter. Consequently, acetonitrile was added to the running buffer to facilitate the elution of hydrophobic PAHs. In the absence of micelles, the separation mechanism was attributed to the solvophobic association of the PAH molecules with hydrophobic chains of the DOSS surfactant and there was a linear correlation between log k (retention factor) and the double bond number of the PAH. However, separations performed in an optimized buffer containing both DOSS and acetonitrile were not able to provide satisfactory performance. The separation of the 16 PAHs was then appreciably improved by adding urea to the running buffer to widen the separation window. At a high sample loading with UV detection, except for benzo[a]pyrene, benzo[b]fluoranthene and benzo[k]fluoranthene, all other PAHs were practically baseline‐resolved using an optimized running buffer containing 50 mM DOSS, 35% acetonitrile and 5 M urea in 8–10 mM borate, pH 9. Laser‐induced fluorescence permitted a very low sample loading and under such a running condition, a baseline resolution was obtained for these three most difficult PAHs. The addition of urea at this level, however, exhibited a noticeable quenching effect on the PAH fluorescent signal.
Proteins of rat serum: I. Establishing a reference two‐dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography‐electrospray mass spectrometry
Authors: Haynes Paul , Miller Ingrid , Aebersold Ruedi , Gemeiner Manfred , Eberini Ivano , Lovati Maria Rosa , Manzoni Cristina , Vignati Mara , Gianazza Elisabetta
Published: May 2005

In the present investigation, we have identified 56 major spots, or spot rows, corresponding to 22 proteins, in the 2‐DE pattern of adult male rats. This was done mainly by applying two complementary techniques, namely immunoblotting and high performance liquid chromatography‐mass spectrometry (HPLC‐MS) peptide mapping. Glycoproteins were characterized by affinity blotting with six lectins. We have also detailed how rat serum differs from human serum in two main respects: (i) relative abundance of individual proteins, which amounts in some cases to a complete absence in either sample, and (ii) varying molecular parameters for homologous proteins. It was thus possible to establish a first‐generation reference map of rat serum proteins, which can be accessed through http://weber.u.washington.edu/∼ruedilab/aebersold.html. We hope the present database will be a useful reference for the evaluation of changes in serum protein distribution in the course of pharmacological and toxicological studies. The recognition of species‐specific proteins appears of special relevance in this respect.
Agarose gel structure using atomic force microscopy: Gel concentration and ionic strength effects
Authors: Maaloum Mounir , Pernodet Nadine , Tinland Bernard
Published: May 2005

Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the “unperturbed” gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogenous with decreasing Tris‐borate‐EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.
Recent developments in preparative free flow isoelectric focusing
Authors: Weber Gerhard , Boček Petr
Published: May 2005

Continuous flow electrophoresis (CFE) is a promising method for preparative fractionation of a variety of biological species, ranging from peptides and proteins to subcellular particles and cells. The high separation efficiency of FFE may be deteriorated by hydrodynamic distortion of zones due to the omnipresent parabolic laminar flow profile. We show in this paper that the detrimental hydrodynamic distortion of separated proteins zones can be reduced, with resultant enhancement of separation efficiency, by employing continuous isoelectric focusing in pH gradients as the actual working regime in an advanced instrumentation. Newly developed media for fast generation of narrow‐ or broad‐range pH gradients under CFE conditions are described. The separation efficiency of these pH gradients is comparable to that of the gradients formed with the aid of synthetic carrier ampholytes. The new media are defined mixtures of nontoxic chemicals, and thus they are compatible with the requirements of human medicine. Experimental data are given showing that the new media offer fractionation of isoforms of proteins, that they offer resolution of proteins differing in isoelectric point (pI) by less than 0.05 pH units, and that these media inhibit proteins precipitation in experiments with human serum proteins.
Finding a universal low‐viscosity polymer for DNA separation
Authors: Heller Christoph
Published: May 2005

We have investigated the viscosity of different commercially available polymers in solution and found that dextran has a low viscosity compared to other polymers of comparable molecular weight and resolving power. This makes it a potentially useful matrix for DNA separation in capillary electrophoresis, where either short time or low pressure are preferred for matrix replacement. We showed that dextran performs well for the separation of oligonucleotides and double‐stranded DNA fragments. Together with the well‐known application for protein separation, this makes dextran a universal polymer for the separation of biological macromolecules.
Proteome and proteomics: New technologies, new concepts, and new words
Authors: Anderson N. Leigh , Anderson Norman G.
Published: May 2005

The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high‐throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.
Proteome analysis: Biological assay or data archive?
Authors: Haynes Paul A. , Gygi Steven P. , Figeys Daniel , Aebersold Ruedi
Published: May 2005

In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.
The Australian proteome analysis facility (APAF): Assembling large scale proteomics through integration and automation
Authors: Walsh Bradley J. , Molloy Mark P. , Williams Keith L.
Published: May 2005

The field of proteomics opens new possibilities for the mass screening of proteins from many different sources. While genomics is well understood to be a big science field, proteomics is just emerging as such. This paper describes the setting up of the first national proteomics facility. The facility has been funded by the Australian government and this funding has allowed the design of purpose built, integrated laboratories with state of the art equipment for large scale proteome research.
New insights into cyclosporine A nephrotoxicity by proteome analysis
Authors: Aicher Lothar , Wahl Daniel , Arce Agustin , Grenet Olivier , Steiner Sandra
Published: May 2005

Using two‐dimensional gel electrophoresis (2‐DE), we recently discovered an association between decreased calcium‐binding protein, calbindin‐D 28 kDa, urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney. This observation prompted us to investigate kidney tissues of other species, including man. In this paper we show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)‐mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in CsA‐treated human kidney‐transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin‐D 28 kDa protein level was found in most of the kidney biopsy sections. The present results strongly suggest that calbindin is a marker for CsA‐nephrotoxicity. The discovery of calbindin‐D 28 kDa being involved in CsA toxicity has evolved from the application of 2‐DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology. Considering the current improvements in proteome methods it is expected that high throughput proteomics will become an indispensable tool in preclinical safety testing.
Charge heterogeneity of macrophage migration inhibitory factor (MIF) in human liver and breast tissue
Authors: Magi Barbara , Bini Luca , Liberatori Sabrina , Marzocchi Barbara , Raggiaschi Roberto , Arcuri Felice , Tripodi Sergio A. , Cintorino Marcella , Tosi Piero , Pallini Vitaliano
Published: May 2005

Macrophage migration inhibitory factor (MIF) is an ubiquitous protein playing various immunological and hormonal roles. Theoretical electrophoretic coordinates calculated from protein sequence in the SWISS‐PROT database (AC P14174) are 12 kDa and pI 8.24. Using two‐dimensional (2‐D) immunoblotting, we have detected isoelectric forms at ca. 11.9 kDa, with pI values of 7.8 and 6.98 in human liver tissue, breast tissue and a cell line and in preparations of human MIF expressed in E. coli. This evidence suggests that MIF charge heterogeneity originates from a post‐translational modification not requiring euka‐ryote‐specific enzymes. We have also detected in human liver a minor immunoreactive spot at pI 6.23, which coincides with the MIF spot in the liver map in SWISS‐2DPAGE. The pI 6.23 isoform also conceivably derives from post‐translational modification, as MIF is known to be encoded in the human genome by a single copy gene.
Micellar electrokinetic chromatography: A convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity
Authors: Viglio Simona , Zanaboni Giuseppe , Cetta Giuseppe , Iadarola Paolo , Luisetti Maurizio , Guglielminetti Maria
Published: May 2005

High performance capillary electrophoresis (HPCE) has been exploited as an analytical method alternative to current procedures for the determination of proteolytic activity of elastases from different sources. Due to some drawbacks with capillary zone electrophoresis (CZE), the mode of operation employed for the assay of elastolytic activity was micellar electrokinetic chromatography (MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide substrate from products of proteolytic reaction was easily achieved in a fused‐silica capillary of 50 cm effective length × 75 μm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters Km and kcat of the proteases investigated. A comparison of these data with those obtained from high performance liquid chromatography (HPLC)‐based experiments showed that MEKC is a convenient technique for studying protease kinetics.
Chiral separation of α‐amino acids by ligand‐exchange capillary electrophoresis using N‐(2‐hydroxy‐octyl)‐L‐4‐hydroxyproline as a selector
Authors: Végvári Ákos , Kilár Ferenc , Schmid Martin G. , Gübitz Gerald
Published: May 2005

The direct chiral resolution of underivatized α‐amino acids by capillary zone electrophoresis (CZE) based on the principle of ligand exchange is described. An N‐(2‐hydroxyoctyl)‐L‐4‐hydroxyproline/Cu(II) complex was used as a chiral selector. Besides amino acids containing aromatic residues, the basic amino acid histidine was resolved. Baseline separations were obtained for all amino acids investigated. The influence of selector concentration, electrolyte composition and pH on the resolution was investigated. It was found that there is a correlation between pI of the amino acids and the optimal pH.
Nonaqueous capillary electrophoresis — its applicability in the analysis of food, pharmaceuticals and biological fluids
Authors: Bjørnsdottir Inga , Tjørnelund Jette , Hansen Steen Honoré
Published: May 2005

The use of nonaqueous electrophoresis media for the application of capillary electrophoresis in the analysis of food, pharmaceuticals and biological fluids is reviewed. Some of the applications are discussed in detail and the benefits of using nonaqueous media in these cases are outlined. Three new applications within pharmaceutical analyses are presented. In these methods either a simple sample pretreatment by dilution with methanol (determination of chlorhexidine in a cream) or selective on‐line capillary electrophoresis mass spectrometry (methods for identification of seizure drugs or opium alkaloids) are used. The choice of organic solvents and electrolytes for nonaqueous capillary electrophoresis are discussed. Furthermore, validation data obtained using capillary electrophoresis based on the nonaqueous principle are listed and discussed.
Nonaqueous capillary zone electrophoresis for separation of free fatty acids with indirect fluorescence detection
Authors: Wang Tianlin , Wei Hongping , Li Sam F. Y.
Published: May 2005

The feasibility of combining nonaqueous capillary zone electrophoresis with indirect fluorescence detection was studied for the efficient separation and sensitive detection of ionizable hydrophobic substances which do not possess practically suitable detection properties. Medium‐ and long‐chain free fatty acids, C6C24, were selected as test compounds. The results showed that such a wide range of medium‐ and long‐chain free fatty acids could be separated between any two consecutive homologs in one run and be detected at a level of about 0.01–0.02 mM in highly basic methanol/acetonitrile media containing fluorescein as coion of background electrolyte for indirect fluorescence detection.
Electrochemical detection in capillary electrophoresis with dual‐parallel on‐capillary electrodes
Authors: Voegel Phillip D. , Baldwin Richard P.
Published: May 2005

A new approach for dual electrode electrochemical detection in capillary electrophoresis (CEEC) is described. In this approach, two identical capillaries, each containing an on‐capillary electrode incorporated permanently onto its tip, were paired together for simultaneous sample injection and detection. This procedure permitted dual‐parallel detection to be performed without the need for painstaking alignment of the electrodes with respect to one another and to the capillary outlet as is required for the off‐capillary microelectrode systems usually employed in CEEC. As a result, independent detection at two electrodes held at different potentials or at two electrodes of different composition or structure could be performed simply and with wide flexibility. Fabrication of on‐capillary electrodes was carried out by sputter‐coating the exit end of the capillaries with a thin layer of Au or Pt. Dual electrode system performance was demonstrated by separation and analysis of phenol and catechol samples. In addition, the detection system was coupled with glucose oxidase for the selective detection of glucose.
Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins
Authors: Rathore Anurag S. , Horváth Csaba
Published: May 2005

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including α‐chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and β‐lactoglobulins A and B, was altered by adding 6‐monodeoxy‐6‐monoamino‐β‐cyclodextrin or carboxymethylated β‐cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6‐di‐O‐methyl)‐β‐cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of β‐cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.
Assay of trypsin activity by capillary isoelectric focusing with laser‐induced fluorescence detection
Authors: Shimura Kiyohito , Kasai Ken‐ichi , Matsumoto Hiroyuki
Published: May 2005

Capillary isoelectric focusing is a highly effective method for the separation of proteins due to focusing as a function of their pI values in the separation process. This technique is also effective for certain types of peptides that focus well. Fluorescence labeling and subsequent detection by laser‐induced fluorescence farther enhance the sensitivity of this technique. This paper demonstrates the utility of this technique in an enzyme assay. A synthetic nona peptide, H‐Gly‐Cys‐His‐Glu‐Ala‐Arg‐Ala‐Glu‐Glu‐OH, was labeled with an iodoacetyl derivative of Lissamine rhodamine B at the thiol group of the cysteine residue as a substrate for trypsin. Trypsin catalyzed the cleavage of the Arg‐Ala bond of the labeled substrate, which focused at pH 4.8, and liberated a shortened, labeled product, H‐Gly‐*Cys‐His‐Glu‐Ala‐Arg‐OH that focused at pH 6.9 (*indicates the label). The product peptide at 3–300 pM was determined with a relative standard deviation of 5.5% (n = 5) by fluorescence detection at 590 nm with excitation by a green line of He‐Ne laser. Incubation of trypsin with the substrate for 10 min at 37°C allowed the determination of 50–250 pg of trypsin, with a relative standard deviation of 5.3% (n = 5).
Protein profile characterization of bacterial lysates by capillary electrophoresis
Authors: Kustos Ildikó , Kocsis Béla , Kerepesi Ildikó , Kilár Ferenc
Published: May 2005

A fast and reproducible method was developed to characterize cell lysates by their electrophoretic profiles using capillary electrophoresis (CE). Characteristic and reproducible patterns were recorded for each bacterial strains when “dynamic sieving” CE, using a polymer solution in the capillary, was applied to distinguish four strains of the Enterobacteriaceae family. The electropherograms showed distinct differences when comparing them to the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) protein profiles. This is certainly a result of the differences in the separation principles and in the detection methods of the two techniques.
A comparison of liver protein changes in mice and hamsters treated with the peroxisome proliferator Wy‐14,643
Authors: Giometti Carol S. , Tollaksen Sandra L. , Liang Xiaoli , Cunningham Michael L.
Published: May 2005

Interspecies differences in the liver response to Wy‐14,643, a potent peroxisome proliferator in rats and mice, have been demonstrated. While both rats and mice show dramatic increases in the number of peroxisomes, the activity of peroxisomal enzymes involved in the β‐oxidation of fatty acids, and heptocyte replication, Syrian hamsters have a more moderate peroxisome proliferation response and no sustained increase in cell replication. Rats and mice, but not hamsters, develop hepatocellular carcinoma after prolonged exposure to Wy‐14,643. To further characterize this species difference, two‐dimensional gel electrophoresis (2‐DE) has been used to compare the effect of 14‐day exposure to various dietary concentrations of Wy‐14,643 on liver protein expression in male mice and hamsters. Digitized images of the 2‐DE protein maps were searched for significant changes. The peroxisome bifunctional enzyme (PBE) enoyl CoA hydratase/3‐hydroxyacyl dehydrogenase, which migrates to the same position in mouse and hamster liver protein 2‐DE patterns, increased in abundance by more than three times the control level in both mice and hamsters. In addition to the quantitative change in PBE, significant quantitative changes (P < 0.001) were found in 49 mouse liver proteins (47 decreasing and 2 increasing) and in 35 hamster liver proteins (27 decreasing and 8 increasing). There was little overlap in the mouse and hamster proteins showing quantitative changes in response to Wy‐14,643, with the exception of PBE and one unidentified liver protein with an approximate molecular weight of 50 000. These results show that although peroxisome proliferation occurs in the livers of both mice and hamsters exposed to Wy‐14,643, other species‐specific changes in proteins occur that are independent of the peroxisome proliferation response and that could be related to species‐specific susceptibility or resistance to liver tumor induction.
Characterization of sugar chain structures of human α‐fetoprotein by lectin affinity electrophoresis
Authors: Taketa Kazuhisa
Published: May 2005

α‐Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex‐type oligosaccharides. Serum AFP samples from cord blood on full‐term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two‐dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above‐mentioned AFP samples. Known specificities of the lectins against complex‐type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.
Analysis of starch structure using fluorophore‐assisted carbohydrate electrophoresis
Authors: Morell Mathew K. , Samuel Michael S. , O'Shea Michael G.
Published: May 2005

The analysis of the fine structure of starches is important to the investigation of linkages between starch structure and function and to the investigation of the properties and roles of starch biosynthetic, modifying and degradation enzymes. Fluorophore‐assisted carbohydrate electrophoresis has recently been introduced as a method for the analysis of the oligosaccharide populations released by the enzymatic digestion of starches, which has advantages in resolution and sensitivity over previously used methods, and provides the capacity for the facile analysis of oligosaccharide populations on either a molar or mass basis. The use of fluorophore‐assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of labeling and separation techniques. Examples of separations using slab gel electrophoresis, DNA sequencer analysis and capillary electrophoresis are presented and we conclude that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100. Examples of isoamylase‐debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through the analysis of oligosaccharides released by the debranching of limit dextrins derived from starches and glycogens is introduced as a useful diagnostic of starch structure. The potential for future development of novel diagnostics for starch structure using fluorophore‐assisted carbohydrate electrophoresis is discussed.
Preparative separaton of plasmid and bacterial artificial chromosome DNA by density gradient electrophoresis in the presence of linear polymers
Authors: Cole Kenneth D.
Published: May 2005

A density gradient apparatus was used to examine the separation of different physical forms and sizes of DNA. A gradient of sucrose was used to stabilize thermal convection during electrophoresis in the column (2.2 cm in diameter). Linear polymers were added to the density gradient and screened for their ability to separate the supercoiled, nicked circular, and linear forms of the plasmid pBR 322. The influence of different concentrations and molecular weights of the polymers was examined on the separation. Polyethylene oxide with a molecular weight of 5 000 000 and a concentration of 0.2% w/v achieved the best separation results for the different physical forms of the plasmid. The order of separation of the different physical forms of the plasmid were linear (fastest), supercoiled, and nicked circular (slowest). These conditions were also used to separate a preparation of bacterial artificial chromosome (BAC) DNA. A rapidly moving form, presumably the supercoiled form, was resolved from a large amount of E. coli genomic DNA and from sheared forms of the BAC DNA.
Capillary electrophoretic separation of 1 to 10 kbp sized dsDNA using poly(ethylene oxide) solutions in the presence of electroosmotic counterflow
Authors: Chen Hsuan‐Shen , Chang Huan‐Tsung
Published: May 2005

DNA fragments of 1 to 10 kbp in length were separated by capillary electrophoresis (CE), using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow. The technique requires filling the capillary with the polymer solution by means of electroosmotic flow (EOF). Separation times of 6–7 min in PEO solutions ranging from 0.3 to 8 × 106 Mr at 375 V/cm were sufficient to separate the 11 components of the dsDNA ladder (0.5 to 10 kbp) by size. The migration behavior of the double‐stranded (ds)DNA fragments, interpreted by “Ferguson plot analysis”, in the system is indistinguishable from that previously reported for capillary zone electrophoresis (CZE) in a polyacrylamide solution without EOF. Potential advantages of conducting CZE using polymer solutions in the presence of EOF are: (i) Possibility of long migration times on short columns; (ii) possibility of introducing relatively viscous, high Mr polymer solutions into narrow capillaries; (iii) possibility of establishing polymer concentration gradients in capillaries; (iv) possibility of concentrating the starting zone by balancing electrophoretic migration and electroosmotic transport.
Thermal degradation and flammability of cellulosics
Authors: Košík M. , Luzáková V. , Reiser V. , Blažej A.
Published: May 2005

The course of pyrolysis, thermooxidation, flame combustion and glowing combustion has been observed with differently treated cellulose samples by means of thermoanalytical methods. The impregnation of cellulose materials by inorganic salts produces a less combustible material, less stable at relatively low temperatures, yet giving more of the solid residue resistant to pyrolysis and combustion processes. The more thermally stable cellulose derivatives, but with insufficient resistance to flame and particularly glowing combustion, have been obtained by cyanoethylation and grafting of acrylonitrile on to cellulose.
Flame‐resistant fibres and fabrics a review of the work at lambeg industrial research association
Authors: Hill B. J.
Published: May 2005

This paper discusses the relative merits of a number of flame‐resistant fabrics. The information quoted draws heavily from a five year programme of research carried out on the subject by Lambeg Industrial Research Association. The fabrics concerned were made either from flame‐resistant fibres or by flame‐resistant treatment of fabrics. The textile problems which can arise in the production of these fabrics are considered and means of avoiding them suggested. Test methods for flammability are discussed briefly and the subject of toxic gases generated from flame‐resistant materials in fire situations is introduced as an area which needs further study. The paper is essentially a situation report describing the present state of knowledge indicating gaps therein, the limitations of the fibres, fabrics and finishes available and hence areas for future work.
Measurement of smoke in fires—a review. I —characteristics of smoke
Authors: Maries K.
Published: May 2005

The Complexity of the behavior of smoke at fires can lead to great difficulties in obtaining measurements from instruments which are both reliable and correlatable with human observations. The first article in this two‐part review considers the principal characteristics of smoke, the factors which influence its behaviour at fires, the basic theory of light transmission through smoke, and the behaviour of the eye in smoky conditions. This information will be used in the second article to assess the performance and limitations of various types of smoke measuring instruments.
The effect of the carbon‐bromine bond type on the fire retardation of unsaturated polyester
Authors: Ravey M. , Fischler Th. , Kramer M.
Published: May 2005

Limiting Oxygen Indexes were determined for a polyester resin fire retarded with a number of different brominated hydrocarbons. It was found that up to a concentration of 28 phr bromine the values obtained were linearly related to the bromine concentration and independent of the carbon‐bromine bond.
The effect of the carbon–bromine bond type on the limiting oxygen index. II—Polymethylmethacrylate and polystyrene
Authors: Ravey M. , Fischler Th.
Published: May 2005

Further evidence has been obtained to suggest that the fire retardation efficiencies of several organo‐bromine compounds, as measured by the limiting oxygen index method, are independent of the type of carbon‐bromine bond, depending almost entirely upon the bromine concentration. Linear regression analysis has confirmed the linearity of the relationship between the limiting oxygen index method and the bromine concentration. The fire retardation efficiency (as measured by the limiting oxygen index method) of the organo‐bromine compounds does, however, depend to a marked extent upon the substrate.