bluuebunny/crossref_metadata_2025 huggingface dataset:
Group‐specific component subtyping in semen and seminal stains by conventional isoelectric focusing
Authors: Kido Akira , Kimura Yukiko , Oya Masakazu
Published: May 2005

Group‐specific component (GC) subtyping in semen and seminal stains was carried out using isoelectric focusing in carrier ampholyte‐generated pH gradients and immunoblotting. In serum samples the anodal bands of GC 1F and of GC 1S disappeared by neuraminidase treatment, but in semen samples these bands remained unchanged after such treatment. The GC 2 type in semen exhibited two bands: the main GC 2 band and another fast band which focused at the position of the cathodic band of GC 1F. These seminal GC bands were unaffected by enzyme digestion. Reliable subtyping was possible in seminal stains stored at 4°C for up to 10 weeks, at room temperature for up to 8 weeks, and at 37°C for up to 5 weeks. The GC subtyping by conventional isoelectric focusing after neuraminidase treatment is simple, economical and useful in medicolegal examination of seminal stains.
Two‐dimensional electrophoretic analysis of down‐regulated proteins in human fibroblasts immortalized by treatment with either 4‐nitroquinoline 1‐oxide or 60Co gamma rays
Authors: Kondo Tadashi , Mihara Koichiro , Inoue Yusuke , Iijima Mikio , Namba Masayoshi
Published: May 2005

Cellular proteins were examined by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in order to determine the mechanisms of cellular aging and immortalization of human fibroblasts. A total of 10 cell lines were studied, four of which were immortalized. OUMS‐24F and SUSM‐1 cells were immortalized by repeated treatment with 4‐nitroquinoline 1‐oxide (4NQO), KMST‐6 cells were immortalized by intermittent exposure to 60Co‐gamma rays, and IMR‐90/SV40 cells were immortalized with simian virus 40 (SV40). None of these immortalized cells were tumorigenic. Four normal human fibroblast cell lines, OUMS‐24, AD 387, KMS‐6 and IMR‐90, and two human cervical cancer cell lines, HeLa and A‐431, were also studied. Applying 2‐D PAGE, the down‐regulation of the identical proteins was observed in the four immortalized cell lines and in the two human cancer cell lines. Their molecular masses were about 80 kDa and the isoelectric points ranged between 5.5 and 6.3. We could not find any information on our proteins from the recent protein data bases. Further characterization of the proteins indicated that they might be phosphorylated cytoskeletal proteins.
Matrix‐assisted laser‐desorption/ionization mass spectrometric approaches for the identification of gel‐separated proteins in the 5–50 pmol range
Authors: Patterson Scott D.
Published: May 2005

The ability to identify and characterize low picomole quantities of gel‐separated proteins has greatly benefited from recent advancements in mass spectrometric analysis methods, particularly peptide‐mass search routines. We are investigating the use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS)) to gain as much mass information as possible from a single gel‐separated protein species. This report details results obtained from one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separations, under nonreducing conditions, of known quantities of three proteins, followed by blotting to Immobilon‐CD. Three methods were used to obtain MALDI‐MS data from a single blotted protein band: (i) direct MALDI‐MS of ∼10% of the band, (ii) cyanogen bromide (CNBr) cleavage of another ∼10% of the band, and (iii) enzymatic (endoproteinase Lys‐C) digestion of the remaining ∼70–80% of the band followed by MALDI‐MS. At the level of 50 pmol of protein loaded onto the gel, data was obtained from all three approaches. At levels down to 5 pmol of protein loaded onto the gel, MALDI‐MS data was obtained from the latter two methods, CNBr and Lys‐C digestions, but not direct MALDI‐MS. Sufficient peptide masses were obtained from the 5 pmol loads to identify two of the three test proteins using four mass search programs. Only limited peptide mass data was obtained from fetuin, a sialylated glycoprotein with six disulfides and no methionines, but it was identified.
Nonreducing two‐dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins
Authors: Reid Gavin E. , Ji Hong , Eddes James S. , Moritz Robert L. , Simpson Richard J.
Published: May 2005

Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two‐dimensional polyacrylamide gel electrophoresis (2‐DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized‐pH‐gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2‐DE gels for performing nonreducing/non‐urea 2‐DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X‐100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide‐mass fingerprinting for identifying 2‐DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2‐DE gel images reported in this study, along with our master 2‐DE gel protein database for both normal human colonic crypts and several colon‐cancer‐derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2‐DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http://www.ludwig.edu.au).
A Saccharomyces cerevisiae Internet protein resource now available
Authors: Latter Gerald I. , Boutell Thomas , Monardo Patrick J. , Kobayashi Ryuji , Futcher Bruce , McLaughlin Calvin S. , Garrels James I.
Published: May 2005

The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two‐dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World‐Wide Web (WWW) server (http://siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS‐PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other.
Phenotyping of apolipoprotein E using immobilized pH gradient gels for one‐dimensional and two‐dimensional separations
Authors: Golaz Olivier , Sanchez Jean‐Charles , James Richard W. , Hochstrasser Denis F.
Published: May 2005

Apolipoprotein E (apo E) is a normal component of several classes of plasma lipoproteins. Apo E phenotypes are closely related to total cholesterol, low density lipoprotein (LDL)‐cholesterol and apo B concentration. The apo E 2/2 phenotype is related to the type III hyperlipoproteinemia due to the defective binding of apo E‐2 to the hepatic receptors. The apo E 4/4 phenotype has been reported to be present in most elderly people suffering from the Alzheimer disease, and is associated with increased risk of coronary heart disease and Creutzfeld‐Jakob disease. Therefore, apo E phenotyping is essential. The method described here uses a precast immobilized pH gradient, avoids time‐consuming separation of lipoproteins from plasma, needs no pretreatment with neuraminidase and involves highly sensitive enhanced chemiluminescence for visualization. Therefore it has many advantages over previously published methods.
Two‐dimensional electrophoresis for the study of blood/serum proteins of the otter, an endangered species
Authors: Miller Ingrid , Gutleb Arno C. , Gemeiner Manfred
Published: May 2005

Studies on serum/blood samples from European otters (Lutra lutra), a species under protection in most countries, were undertaken. Building of a two‐dimensional electrophoresis (2‐DE) protein identification map was started, identifying serum proteins by pattern comparison with cat or human serum protein maps and by immunoblotting using cross‐reacting antibodies against the respective human serum proteins. The capture of endangered species is very restricted, thus making fresh samples almost unavailable; most investigations therefore had to be performed on samples collected from animals found dead. Patterns obtained from specimens of this source were compared to the established pattern of serum proteins and of blood or liver cells. Some of the major differences observed can be explained by contamination with cellular proteins and/or the onset of lytic processes generating breakdown products, as shown by preliminary in vitro experiments. Better knowledge of “normal” serum protein patterns and interfering products will facilitate interpretation of data collected from samples with a less defined history.
Spermatocytes and round spermatids of rat testis: Protein patterns
Authors: Cossio Gabriela , Sanchez Jean‐Charles , Golaz Olivier , Wettstein Rodolfo , Hochstrasser Denis F.
Published: May 2005

Spermatogenesis is a process in the testis that involves meiotic cell division and spermiogenesis. The mechanisms of regulation and its associated proteins are mostly unknown. This publication shows the two‐dimensional (2‐D) gel electrophoresis protein map obtained from rat testis using nonlinear 3.5–10 immobilized pH gradients for the first‐dimensional separation. Eighteen proteins were successfully identified in the SWISS‐PROT protein database using amino acid analysis of proteins recovered from polyvinylidene difluoride (PVDF) membranes and verified for one of them by comparison with Anderson's rat liver reference map. Fourteen new polypeptides were identified and four were previously known. Two of these new proteins were closely related to the spermatogenetic process. T‐complex protein 1 is expressed in large amounts in germ cells. Androgen‐dependent sperm‐coating glycoprotein is secreted by epididymal cells. In order to detect changes in protein expression during meiosis and spermiogenesis, spermatocytes and round spermatid cell populations were purified by centrifugal elutriation and compared. In this way several proteins not found in the spermatocyte 2‐D images could be highlighted. The sperm‐coating glycoprotein was thus shown to be present in large amounts in round spermatids.
Long‐term induction of an aldose reductase protein by basic fibroblast growth factor in rat astrocytes in vitro
Authors: Laeng Pascal , Bouillon Philippe , Taupenot Laurent , Labourdette Gérard
Published: May 2005

Basic fibroblast growth factor (bFGF) is known to elicit various developmental‐like effects on astrocytes in vitro, but these effects were studied mainly over short‐term periods. In this work we asked the question whether bFGF could induce long‐term effects on rat astrocytes in culture. This factor was found to induce only a transient mitogenic effect lasting less than 48 h, even when the treatment was carried on for 4 days. By contrast, it induced long‐term effects on the rate of synthesis of several proteins as seen by two‐dimensional polyacrylamide gel electrophoresis after labeling the cells with [35S]methionine. The most upregulated protein was extracted from preparative gels of soluble extracts of cultured bFGF‐treated astrocytes and of normal brain. It was characterized by internal amino acid microsequencing. Two tryptic digest peptides had N‐terminal sequences similar to rat lens aldose reductase. This protein was also expressed in oligodendroglial and neuronal cells in culture, but it was not upregulated by bFGF. Aldose reductase is thought to be involved in a minor pathway of glucose metabolism and in diabetic complications. Its longterm regulation by bFGF will possibly help in the understanding of its actual physiological role.
Programmed cell death in sympathetic neurons: A study by two‐dimensional polyacrylamide gel electrophoresis using computer image analysis
Authors: Amess Bob , Tolkovsky Aviva M.
Published: May 2005

The technique of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled with computer image analysis was used in this study to examine changes in protein expression occurring during the onset of programmed cell death (PCD) in rat sympathetic neurons following withdrawal of nerve growth factor (NGF). Sympathetic neurons from superior cervical ganglia of postnatal day‐one Wistar rats were cultured in the presence of NGF for 24 h and then either maintained in the presence of NGF or deprived of NGF for a period of 8 h. To label the proteins being synthesised, neurons were cultured in the presence of L‐[35S]methionine for a further 2 h under the same conditions but with 3% of the normal methionine concentration. Neuronal proteins were then analysed by 2‐D PAGE using immobilised pH gradient (IPG) gel strips in the first dimension. For the second dimension a custom‐built electrophoresis system capable of running multiple sodium dodecyl sulfate (SDS)‐PAGE slab gels in a vertical configuration, with good temperature control (± 0.7°C) was used and is described in this paper. Proteins resolved on the dried gels were visualised using storage phosphor technology and the digitised images subjected to rigorous analysis using the QUEST II software system. Seventeen proteins whose relative synthesis decreased and four proteins that increased upon NGF withdrawal were located and are documented.
Two‐dimensional electrophoresis of thylakoid protein patterns in two wheat cultivars with different sensitivity to sulfur dioxide
Authors: Ranieri Annamaria , Castagna Antonella , Bini Luca , Soldatini Gian Franco
Published: May 2005

Plants of two wheat cultivars (Triticum aestivum L., cultivars Mec and Chiarano), differing in sensitivity towards sulfur dioxide, were fumigated with 96 nL L−1 of SO2 for four months. Thylakoid membranes were subjected to two‐dimensional electrophoresis in order to detect possible changes in the protein patterns. We found the greatest alterations following treatment in the polypeptide profiles of the most sensitive cultivar, Mec, even if some significant changes were also detected in the plants of the cultivar Chiarano.
A comparison of resolution of DNA fragments between agarose gel and capillary zone electrophoresis in agarose solutions
Authors: Weiss George H. , Garner Mark , Yarmola Elena , Boček Petr , Chrambach Andreas
Published: May 2005

The resolving power of capillary zone electrophoresis (CZE) is compared to that of gel electrophoresis (GE) under similar conditions (agarose, similar length of DNA fragments, identical buffer) but with differences in temperature and field strength. The comparison is based on the time required to reach a desired degree of resolution by each of the two methods. A resolution parameter is developed which is equally applicable to CZE, with relatively diffuse initial conditions in the absence of stacking and measurements expressed in terms of time, and to GE, in which measurements are expressed in terms of spatial parameters. The resolution time in CZE using agarose solutions at 40°C was found to be greater by at least one order of magnitude than that in GE using agarose gels. Thus, the increased migration velocity due to high field strength in CZE substantially outweights the lower dispersion in GE.
Some reminiscences of the history of electrophoresis
Authors: Rilbe Harry
Published: May 2005

Dole's and Longsworth's solution of the whole set of moving boundary equations by using the dimensionless transference numbers is reviewed. Longsworth's teachings on the nature of transference numbers and his pondering over transference numbers of proteins led the author to inquire why transference numbers of ampholytes were never measured. Logical reasoning on this subject led to the invention of isolectric focusing.
From image processing to classification: III. Matching patterns by shifting and stretching
Authors: Skovgaard Ib Michael , Jensen Kirsten , Søndergaard Ib
Published: May 2005

A method for the classification of electrophoretic patterns is described and tested on a data set representing ten wheat varieties. The method attempts to match each electropherogram to each variety by a transformation involving displacement and stretching along the x‐axis. This is done essentially by the method of least squares, which uses only the information contained in the electropherogram itself to adjust it to the variety in question. The method is completely automatic and works extremely well by classifying 98% of the spectra correctly, judged by cross validation.
Chemical stability of polyacrylamide‐coating on fused silica capillary
Authors: Nakatani Manabu , Shibukawa Akimasa , Nakagawa Terumichi
Published: May 2005

The chemical stability of polyacrylamide coatings on a fused silica capillary for capillary electrophoresis was investigated with respect to temperature of capillary and pH as well as salt concentration of running buffer solution by evaluating their effects on electroosmotic mobility. The capillary covalently bonded to linear polyacrylamide through Si‐C linkage was stable in buffer solutions of pH 2.3–8.0 at 30°C for 30 days, whereas the capillary bonded through Si‐O‐Si‐C linkage was damaged at pH above 4.6. The electroosmotic mobility observed in both capillaries increased with increasing temperature, though the degree of increase was smaller in the Si‐C linked capillary than in the Si‐O‐Si‐C linked capillary. The increase in the buffer concentration resulted in increased electroosmotic mobility in the Si‐O‐Si‐C linked capillary, but no effect was observed for the Si‐C linked capillary.
Effect of organic modifier concentrations on electrokinetic migrations in micellar electrokinetic chromatography
Authors: Chen Nong , Terabe Shigeru , Nakagawa Terumichi
Published: May 2005

The effect of low concentrations of organic modifiers on the electroosmotic mobility, μeo, and electrophoretic mobility of the micelle, μep, mc, in micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate (SDS) solutions was investigated. The results showed that μeo was mainly influenced by the viscosity of the running solution. A linear relationship between μeo and the concentration of the organic modifier was found in MEKC. A newly defined parameter, the mobility ratio, Rm, mc, which is the ratio of the electro‐phoretic mobility of the micelle to the electroosmotic mobility, and which characterizes the surface charge density of the micelle in MEKC system with organic modifiers, was introduced. A linear relationship between Rm, mc and the organic modifier concentration was observed. The parameter Rm, mc was found to be useful to investigate the changes in the micellar phase with the addition of organic modifier. The measured μep, mc values showed different and interesting characteristics among different organic modifiers.
Fluorescence imaging detection for capillary isoelectric focusing
Authors: Wu Xing‐Zheng , Wu Jiaqi , Pawliszyn Janusz
Published: May 2005

A simple laser‐induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the ultrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long capillary column by a cylindrical lens. Fluorescence emission is detected by a charge‐coupled device (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b‐phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC‐labeled proteins (i) α‐D‐galatosylated FITC‐albumin, (ii) insulin‐FITC, and (iii) casein‐FITC, are used as model samples. Fluorescence images of the model samples are measured during the CIEF process. The focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC‐labeled proteins are at the level of 10−10 M, and the mass detection limits are about 4.5 × 10−17 mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10−11 M or amole (10−18 mole) level protein samples with a band‐pass filter more specific to the fluorescence light. Potential applications of the LIF imaging system in addition to quantitation of separated fluorescent species in various capillary electrophoresis methods can also include investigation of interaction between analytes focused in a capillary column.
Fluorescence‐labeled peptides as isoelectric point (pI) markers in capillary isoelectric focusing with fluorescence detection
Authors: Shimura Kiyohito , Kasai Ken‐Ichi
Published: May 2005

Commercially available peptides, mostly angiotensin derivatives, were labeled at their N‐terminal amino group with 5‐carboxytetramethylrhodamine succinimidyl ester, to obtain fluorescent pI markers for capillary isoelectric focusing with fluorescence detection. The labeled peptides were purified by reversedphase chromatography. They were well separated on isoelectric focusing in a polyacrylamide gel slab and their pIs were determined by comigration with protein‐pI markers. The fluorescent markers could be detected as sharp peaks in capillary isoelectric focusing with laser‐induced fluorescence detection (He‐Ne laser, 1 mW, 543.5 nm). The detection limit was found to be around 3 × 10−12 M (0.8 amol). Tetramethylrhodamine‐labeled pea lectin (3 pg) was subjected to capillary isoelectric focusing and the pIs of the fluorescent derivatives of the lectin were determined by using the fluorescence‐labeled peptides as pI markers.
Further study on the use of uncharged β‐cyclodextrin polymer in capillary electrophoresis: Enantiomeric separation of some α‐hydroxy acids
Authors: Fanali Salvatore , Aturki Zeineb
Published: May 2005

Uncharged β‐cyclodextrin polymer was used as chiral selector for the enantiomeric separation of some α‐hydroxy acids by capillary electrophoresis. Complexation and enantiomeric resolution of mandelic acid, m‐hydroxy and p‐hydroxymandelic acid, 3,4‐dihydroxymandelic acid, as well as 2‐ and 3‐phenyllactic acid were studied, changing the concentration of the β‐cyclodextrin polymer added to the background electrolyte at different pH in the range of 4.5–7. Furthermore, the effects of the concentration of the background electrolyte, column temperature, and applied voltage on chiral resolution were also examined. The best enantiomeric separations were obtained using a background electrolyte at pH 6 containing 100 mg/mL of β‐cyclodextrin polymer.
Repetitive sequence fingerprinting in the long range mapping of mammalian genomes
Authors: Hamvas Renata M. J. , Lehrach Hans R.
Published: May 2005

This review presents some properties of interspersed repeats, particularly human and mouse repeats, and shows how these have been utilized in long‐range genome mapping. The link between the distribution of such repeats and their relationship with genome organization is discussed.
Oligonucleotide (CAC)5 fingerprinting: Validity and reliability in paternity testing
Authors: Papiha Surinder S. , Sertedaki Amalia
Published: May 2005

Following the developments of DNA fingerprinting (Jeffreys et al., Nature 1985, 314, 67–73) using the minisatellite probes 33.15 and 33.6, many other fingerprinting probes have been described. The wide type M13 phage, 3′ HVR region of alpha globin gene, F10 and various synthetic oligonucleotide probes have been shown to produce multiallelic and individual specific DNA fingerprints. The two probes developed by Jeffreys et al. have been extensively used in forensic and paternity determination in various laboratories, however, for the use of DNA fingerprints produced by synthetic oligonucleotide probes, still need critical evaluation. In this paper the statistical evaluations of the fingerprinting probes (CAC)5 was carried out by comparing the results produced by Jeffreys et al., using probes 33.15 and 33.6. The mutation rate calculated for (CAC)5 as 0.015 is nearly similar to the probe 33.15 (0.011) but higher than the probe 33.6 (0.005). The data obtained using (CAC)5 fingerprint in paternity cases from the northeast of England are presented.
Statistical issues in DNA evidence
Authors: Brookfield John F. Y.
Published: May 2005

Some of the statistical issues involved in the interpretation of DNA evidence in courts, particularly focusing on the adversarial process as seen in the UK and the USA, are discussed. The appropriate way to consider the use of DNA evidence in courts is through the use of a likelihood ratio. In this, the relative likelihoods of the DNA evidence are calculated under two hypotheses, respectively that the subject is or is not the source of a DNA sample found at the scene‐of‐crime. This formulation allows, using Bayes' theory, the prior probability of guilt, based on the other evidence in a case, to be incorporated in a formal way into calculation of a final, or posterior, probability of guilt. The most controversial aspect of DNA evidence has concerned the calculation of the probability of a match between an innocent suspect and DNA from the scene‐of‐crime. The calculation is affected by subdivision within the population and the possibility of relatedness between the suspect and the true source of the scene‐of‐crime DNA. The qualitative importance of these effects will be discussed. In addition to the scientific questions concerning the likelihood ratio, there have been a number of other concerns about the use of DNA evidence, and other evidence presented in a statistical form, in courts. These include the question whether accurate or conservative values of the likelihood ratio should be presented, and the abilities of juries to deal with quantitative evidence.
Allele frequency distribution for the variable number of tandem repeat locus D10S28 in Tamil Nadu (South India) population
Authors: Pandian S. Karutha , Kumar Subbaiah , Krishnan Manikkam , Dharmalingam Kuppamuthu , Damodaran Chenniappan
Published: May 2005

Allele frequencies were determined in unrelated individuals of Tamil speaking population from the Madras City (Tamil Nadu, South India) area for the polymorphic DNA locus D10S28 using the probe TBQ7. Membranes hybridized with the probe YNH24 were subjected to deprobing and were subsequently hybridized with random priming – labeled, purified inserts of TBQ7. The sizes of the fragments were grouped to 100 bp as well as to arbitrary fixed bins (Federal Bureau of Investigation/Royal Canadian Mounted Police). There were 14 bins in the latter with the most common bin being 11 (1789–1924 bp) with a frequency of 9.8%. We observed a heterozygosity of 92% comparable to Caucasian populations. The data presented here can be used as the basis for utilizing this variable number of tandem repeats (VNTR) DNA marker for paternity determinations and forensic investigations.
Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis
Authors: Wiese Ulrich , Wulfert Michael , Prusiner Stanley B. , Riesner Detlev
Published: May 2005

Gerstmann‐Sträussler‐Scheinker syndrome (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt‐Jakob disease (CJD) are caused by point mutations or octarepeat insertions in the prion protein (PrP) gene. In the present work a method was established that is appropriate for a thorough screening for mutations in the PrP gene and is generally applicable to screenings of any given gene. Temperature gradient gel electrophoresis (TGGE) was modified at two critical steps by UV cross‐linking of the DNA strands and by replacing the spatial gradient with a temporal one. The shift of a DNA band in temporal temperature gradient gel electrophoresis (tTGGE) due to a mutation can be calculated as a function of the position of the mutation in the sequence. Appropriate DNA fragments were selected for polymerase chain reaction (PCR) amplification and analysis by tTGGE on the basis that the predicted band shifts amount to more than 10% of the migration distance for all possible mutations. The accuracy of the prediction was tested experimentally with ten known mutations in the human PrP gene, and quantitative agreement between theory and experiment was achieved. Thus, this screening method is also a suitable means to verify the absence of mutations in a given gene segment.
Effective ion‐pairing for the separation of basic proteins in capillary electrophoresis
Authors: Okafo George N. , Vinther Anders , Kornfelt Troels , Camilleri Patrick
Published: May 2005

The addition of the sodium salt of phytic acid to the separation buffer (pH's 6.0–9.5) has allowed the analysis of a number of basic proteins (pIs > 9) by capillary electrophoresis. The method of analysis is simple and leads to considerable improvement in peak shape. Some very basic proteins, totally adsorbed onto the capillary fused silica surfaces in the presence of buffer only, can be analysed as sharp signals when this polyanionic species is included in the running electrolyte. These improvements in analysis are thought to arise as a result of the suppression of coulombic interactions between these positively charged proteins (ion‐paired to phytic acid) and the negatively charged silanol groups on the inner wall of the capillary.
Separation of cerebrospinal fluid proteins using capillary electrophoresis: A potential method for the diagnosis of neurological disorders
Authors: Cowdrey Geoffrey , Firth Maria , Firth Gary
Published: May 2005

A method for the analysis of proteins in cerebrospinal fluid (CSF) using free solution capillary electrophoresis (FSCE) was developed, in which the adsorption of proteins to the fused silica capillary wall was virtually eliminated. FSCE was carried out on 30 randomly selected CSF samples. Good sensitivity and resolution was obtained with minimal baseline noise. These preliminary results clearly demonstrated the potential of FSCE to analyse proteins in CSF, both qualitatively and quantitatively more rapidly than has been possible hitherto. A total run time of approximately 30 min allowed between 20 to 25 peaks to be separated. Many peaks with long migration times were detected in CSF that appeared to be specific since they were not detectable in corresponding serum samples. The separation patterns for most of the CSF samples selected showed relatively similar patterns but there were often minor qualitative and quantitative differences. Four CSF samples showed patterns that were significantly different from the others, primarily in the peaks with long migration times. A larger clinical study is now being undertaken.
Training software for electrophoresis
Authors: Reijenga Jetse C. , Martens Johannes H. P. A. , Everaerts Frans M.
Published: May 2005

Computer programs, simulating electrophoretic separations, were evaluated and discussed with respect to their suitability for training purposes. Quite a number of them, mainly those dealing with steady‐state phenomena, are sufficiently fast and user‐friendly for the purpose of visualization of electrophoretic principles. Transient‐state or dynamic models, however, are more suitable for the advanced user, mainly because of their inherent complexity and long calculation times.
Dynamics of peak dispersion in capillary zone electrophoresis including wall adsorption: II. Exact analysis of unsteady linear adsorptive dispersion
Authors: Štědrý Milan , Gaš Bohuslav , Kenndler Ernst
Published: May 2005

An exact analysis of the unsteady axial dispersion of an analyte, undergoing a linear adsorption at the column wall in capillary zone electrophoresis, is presented. A system of partial differential equations – in which the radial coordinate is one of the independent variables – is taken as a model for linear wall adsorption. It is shown that the dispersion is a sum of two terms, one which depends linearly on time and whose exact form is generally known, and a nonlinear one. The most interesting result of this work is that it derives another system of differential equations, which this nonlinear term is to satisfy. It makes it possible to present a closed formula for the asymptotic value of the nonlinear term, i.e., its limit for large time. Its behavior for times close to zero is also studied.
Contribution of capillary coiling to zone dispersion in capillary zone electrophoresis
Authors: Kašička Václav , Prusík Zdeněk , Gaš Bohuslav , Štědrý Milan
Published: May 2005

A mathematical model of the deformation of the analyte zone in capillary zone electrophoresis (CZE) due to capillary coiling is presented and the influence of capillary coiling on the distribution of electric current density inside the capillary is described. The model gives a quantitative description of the potential contribution of capillary coiling to the total zone dispersion in CZE. The zone broadening caused by capillary coiling is calculated as the difference of migration distances of the particles migrating at the inner circumference of the capillary coil and those migrating at the outer circumference. This difference is shown to be directly proportional to the capillary diameter and to the number of capillary coils, i.e. indirectly proportional to the radius of the capillary coils into which the capillary of the given length is coiled. The contribution of capillary coiling to the total zone dispersion is compared with the contribution of the longitudinal diffusion of low and high molecular mass analytes and bioparticles. It is shown that, especially in the case of CZE separation of macromolecules and particles, capillary coiling can significantly decrease the separation efficiency. For that reason the small radius coiling of the capillary column in the CZE apparatuses should be avoided.
Electrophoresis between sieving and reptation: An investigation of the role of shape fluctuations in electrophoresis
Authors: Völkel Armin R. , Noolandi Jaan
Published: May 2005

We present numerical simulation results of electrophoretic mobilities of flexible polyelectrolytes over a wide molecular size range moving through gels with various pore sizes. The data are compared to existing models for different molecular size regimes and to experimental results. We observe rather pronounced shape fluctuations of the polyelectrolytes which, especially for larger gel pores or small molecules, have a strong impact on the dynamics of the molecules. Electrophoretic separation, as is used e.g. for DNA sequencing, is best achieved for polyelectrolytes with a radius of gyration of the order of the average pore radius of the gel, i.e. in a molecular size regime where the polyelectrolyte interacts with only a few gel fibers at a given time. A decrease of the gel pore size leads to a systematic decrease of the electrophoretic mobility, but does not lead to a qualitative change in the molecular size dependence, as long as the pore size is larger than the persistence length of the polyelectrolyte.
A quantitative study on the effect of organic modifiers in micellar electrokinetic chromatography
Authors: Chen Nong , Terabe Shigeru
Published: May 2005

A quantitative study was made on the effect of organic modifiers in micellar electrokinetic chromatography (MEKC). A linear relationship between 1/k′ and organic modifier concentration ([L]) at lower concentration range was derived and experimentally verified. This linearity was also examined at higher concentrations of organic modifier. The results showed that the linear relationship between 1/k′ and [L] can only be observed under the situation that no significant change on micellar structure occurs upon the addition of organic modifiers. The mobility ratio of the solute which characterizes the properties of the solute was found to be mainly independent of [L]. A resolution equation for the neutral solutes was also derived in term of mobility ratio.
The human keratinocyte two‐dimensional gel protein database (update 1995): Mapping components of signal transduction pathways
Authors: Celis Julio E. , Rasmussen Hanne Holm , Gromov Paul , Olsen Eydfinnur , Madsen Peder , Leffers Henrik , Honoré Bent , Dejgaard Kurt , Vorum Henrik , Kristensen Dan Back , Østergaard Morten , Haunsø Anders , Jensen Niels Aagaard , Celis Ariana , Basse Bodil , Lauridsen Jette B. , Ratz Gitte P. , Andersen Annette H. , Walbum Else , Kjærgaard Inge , Andersen Inger , Puype Magda , Van Damme Josef , Vandekerckhove JoëL
Published: May 2005

The master two‐dimensional (2‐D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 none‐quilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post‐translational modifications. 1082 polypeptides have been identified (protein name, organelle components, etc.) using a procedure or a combination of procedures that include (i) comigration with known human proteins, (ii) 2‐D gel immunoblotting using specific antibodies, (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry, (v) vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full‐length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2‐D gel immunoblotting of crude keratinocyte lysates in combination with enhanced chemoluminescence (ECL) detection. Identified proteins are listed both in alphabetical order and with increasing SSP number, together with their Mr, pI, cellular localization and credit to the investigator(s) that aided in the identification. Ultimately, the aim of the comprehensive database is to gather – through a systematic study of ekeratinocytes – qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and to pinpoint signaling pathways and components affected in various skin diseases, cancer included.
Genetic mapping of restriction landmark genomic scanning loci in the mouse
Authors: Okuizumi Hisato , Okazaki Yasushi , Ohsumi Tomoya , Hayashizaki Yoshihide , Plass Christoph , Chapman Verne M.
Published: May 2005

Restriction landmark genomic scanning (RLGS) was originally proposed as a high‐speed method for surveying a large number of restriction landmarks in genomic DNA. The effort to apply this method to genetic analysis has been made, resulting in developing the new approach for the rapid construction of the genetic map of complex mammalian genomes (RLGS spot mapping). Especially, the use of NotI as the restriction landmark for genetic studies suggests that there is a high probability that a significant number of these RLGS loci will be associated with CpG islands of functional genes. Moreover, it is possible to use the RLGS spot mapping to analyze genetic map‐poor species very rapidly for linkage of recessive mutations or segregating traits, because it does not rely upon cloned probes or sequences. In this paper, we summarize the progress that has been made in the practical application of the RLGS method to genetic analysis using congenic strains, recombinant inbred (RI) strains, and in interspecific backcrosses of mice.
A single gel analysis of 575 dominant and codominant restriction landmark genomic scanning loci in mice interspecific backcross progeny
Authors: Okuizumi Hisato , Okazaki Yasushi , Ohsumi Tomoya , Hanami Takeshi , Mizuno Yosuke , Muramatsu Masami , Hayashizaki Yoshihide , Plass Christoph , Chapman Verne M.
Published: May 2005

We identified 575 new NotI landmarks of C57BL/6(B)‐ and, M. spretus (S)‐specific, dominant and codominant loci which were segregated in B × S interspecific backcrosses (BSS), using the restriction landmark genomic scanning (RLGS) spot mapping method. All of these loci were visualized on a single RLGS profile which was produced with NotI‐PvuII‐PstI. These landmarks include 250 newly identified S‐specific spots in addition to the previously reported 325 B‐specific spots. The S‐specific spots were identified by reading full or half intensity, based on the property that the spot intensity of the autoradiographic signal reflected the copy number of an end‐labeled restriction landmark. The cumulative map is 1341 cM and it is based upon 985 meiotic events in 72 backcross progeny. This map covers 90% of the total estimated length of the mouse genetic map. This map provides a good tool for the high‐speed genome scanning assay in the mouse genome by a single RLGS gel analysis.
Analysis of protein synthesis by two‐dimensional gel electrophoresis in T cells persistently infected with coxsackie B virus
Authors: Gimenez Beatriz , Amarasekera Deepthi , Argo Evelyn , Cash Phillip
Published: May 2005

Coxsackie B viruses (CBV) have been implicated in various human diseases that present as either limited acute infections or prolonged chronic infections. A number of investigations have suggested that a virus‐induced immune dysfunction might play a role in in vivo pathogenesis. In the current study, we describe CBV infection of two human T cell‐derived cell lines (Jurkat and MOLT‐4 cells) as potential models for CBV infection of lymphocytes. Short term (up to 144 h post‐infection) CBV infection of either cell line resulted in a decline in the viability of the cell population together with an approximate 10‐fold rise in the titre of infectious virus during the period of incubation. Analyses of the intracellular proteins by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) demonstrated that although putative virus proteins were detectable there was minimal inhibition of the cellular protein synthesis following CBV infection. This contrasted with the more permissive and highly lytic CBV infection of HEp‐2C cells (Cash, Electrophoresis 1991, 10, 793–800). Persistently infected cell lines from both Jurkat and MOLT‐4 cells (piJURKAT‐3673 and piMOLT‐2667 cells) were established. Analyses of intracellular protein synthesis of these persistently infected cell lines showed the synthesis of novel proteins not detected for the corresponding uninfected parental cell line. There were no significant alterations in overall cellular protein synthesis detectable by the small format 2‐D PAGE system employed in these investigations. The data presented in the current investigation will contribute towards studies on virus‐induced responses of specific biological functions associated with T cells.
The use of solid phase concentrators for on‐line preconcentration of metallothionein prior to isoform separation by capillary zone electrophoresis
Authors: Beattie John H. , Self Ron , Richards Mark P.
Published: May 2005

UV absorbance is a convenient detection method for monitoring a wide variety of different components separated by capillary electrophoresis (CE). However, a significant disadvantage of UV detection is its low sensitivity and this is a major factor limiting the application of CE as an analytical technique. In order to improve sensitivity, several methods of on‐line sample preconcentration by electrofocusing, including “stacking” and isotachophoresis, have been investigated and the potential of on‐line solid phase preconcentrators for this purpose has also been demonstrated. We have developed methods for the separation of isoforms of metallothionein (MT), a low Mr metal‐binding protein which functions as a detoxification system and cellular buffer for heavy metals, primarily in liver and kidney. While a variety of modifications to the capillary surface and electrolyte chemistry have been used to resolve many MT isoforms in liver samples from a variety of species, there are few available alternatives to UV detection and thus sensitivity limits the application of these methods to samples with low MT levels, such as urine or plasma. In this study we have investigated the use of an inexpensive and easy to assemble C18 concentrator which was fitted to the capillary inlet to extend the detection limits for MT isoform analysis. Samples were pressure loaded onto the concentrator for up to 5 min, eluted using 33% acetonitrile and then subjected to electrophoresis in borate or phosphate buffer. The purified isoforms MT‐1 and MT‐2 from rabbit liver were best resolved using an acidic acetonitrile eluent and a phosphate buffer electrolyte at pH 7. Sensitivity enhancement was >700‐fold and the relationship between loading time and isoform absorbance peak area was linear when only a small proportion of concentrator capacity was used. Analysis of sheep liver extracts demonstrated that both sensitivity and specificity were enhanced. The potential of other solid‐phase concentrators is discussed.
Nonlinear “Ferguson curves” by two runs of the commercial automated HPGE‐1000 gel electrophoresis apparatus with intermittent scanning of fluorescence
Authors: Yarmola Elena , Chrambach Andreas
Published: May 2005

The exploitation of gel electrophoretic migration distance to gain information of molecular and gel fiber properties depends on the functions relating mobility with gel concentration. To the degree that these are nonlinear, the definition of those functions by past methods has been excessively laborious or, in application of gel concentration gradients, based on a number of assumptions. The recent commercial introduction of gel electrophoresis apparatus capable of intermittent scanning of the pattern promised to solve these problems. The present study shows that such apparatus allows for a precise definition of a nonlinear “Ferguson curve” (mobility vs. gel concentration) in two experiments, using different gel concentrations in the eight channels of the HPGE‐1000 apparatus and 5–29 scans in each during the course of an electrophoretic run. Simultaneously, these Ferguson curves are obtained for five components of a DNA ladder ranging in DNA length from 121 to 1857 bp.
Rapid detection of toxoplasmic nucleic acid by enzyme‐linked immunofiltration‐assay after membrane transfer
Authors: Aubert Dominique , Lepan Herve , Lemaire Pascale , Foudrinier Frederique , Marx‐Chemla Cathy , Bonhomme Annie , Pinon Jean‐Michel
Published: May 2005

We report the use of enzyme‐linked immunofiltration‐assay (ELIFA) for rapid detection of Toxoplasma gondii DNA after transfer to nitrocellulose or nylon membranes. Saturation, specific labeling with a nonradioactive oligonucleotide probe and washing steps are included in an active filtration process, which is controled by microcomputer regulating reagent flow. This method provides excellent specificity, reproducibility and repeatability. Recycling in a closed circuit or by reversing the direction of flow provides the advantage of reducing the required volume of costly reagents (e.g. probes). ELIFA does not require lengthy membrane manipulation, and hybridization as well as stringency and revelation steps can be automated. The procedure can be carried out in less than 2 h, which is a major time saving compared to conventional solid‐phase molecular hybridization techniques.