bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the solvent motion in electrophoretic systems
Authors: Ermakov Sergey V. , Zhukov Michael Y. , Righetti Pier Giorgio 
Published: May 2005

AbstractA corrected model describing transport processes for multicomponent mixtures in electric field is proposed. This model is more consistent compared to the other models of this sort used for simulations of electrophoresis in the case of concentrated solutions. The main idea underlying the model is in accounting for the motion of a solvent. Usually the concentrations of solutes are considered small compared to that of a solvent and the equation describing its motion is not considered. This automatically leads to the violation of momentum balance and, hence, to the defects of the model itself. In the model presented in the paper by means of redefining the mass fluxes the balance of momentum is satisfied automatically while the equations governing the evolution of the system look more symmetric and simple. This model allows us to discover some fine effects in evolution of the mixture and clarify the essence of some conservation laws (conservation of the Kohlrausch function) arising in the simplified mathematical models describing electromigration phenomena.
Northern blot analysis of simple repetitive sequence transcription in plants
Authors: Gortner Götz , Pfenninger Markus , Kahl Günter , Weising Kurt 
Published: May 2005

AbstractThe presence of simple repetitive sequence motifs in RNA from various plant species was probed by Northern blot analysis. Hybridization of total, poly(A)+‐ and poly(A)−‐RNA with microsatellite‐complementary oligonucleotide probes revealed distinct bands with most but not all probe/species combinations, demonstrating the presence of di‐, tri‐ and tetranucleotide repeat motifs in plant transcripts. Only trinucleotide repeat‐derived hybridization signals were found to be enriched in the poly(A)+‐fraction. The quality of Northern blot signals proved to be highly dependent on hybridization stringency. Thus, under the stringency conditions usually applied for oligonucleotide hybridization, some probes [(GT)8, (CAC)5, (TCC)5, and (CCTA)4] cross‐hybridized to bands corresponding in size to 18S and/or 26S rRNA. Cross‐hybridization to rRNA was significantly reduced at higher stringencies. These results stress the importance of carefully adjusting the hybridization conditions in Northern blot analysis of simple sequence transcripts.
(GTG)5 allows the distinction between different isolates of the nematode Caenorhabditis elegans
Authors: Benecke Mark , Epplen Jörg T. , Schierenberg Einhard 
Published: May 2005

AbstractWe have not been able to distinguish different isolates from the nematode Caenorhabditis elegans by morphological means. However, they differ on the molecular level and strains from several geographic regions can be identified with the help of “genetic fingerprints” using the oligonucleotide probe (GTG)5.
Determination of the kinetic parameters of adenosine deaminase by electrophoretically mediated microanalysis
Authors: Saevels Jan , Van Schepdael Ann , Hoogmartens Jos 
Published: May 2005

AbstractThe possibility of determining the Michaelis constant of the irreversible deamination of adenosine to inosine by adenosine deaminase, using capillary electrophoresis, was investigated. This paper describes the use of electrophoretically mediated microanalysis (EMMA) as the technique for carrying out the assay. Initial reaction velocities of the enzymatic reaction were estimated from the peak area of inosine, and the Michaelis constant was calculated according to the Lineweaver‐Burk equation. The result (Km = 5.3 × 10−5 M ± 8 × 10−6 M) was consistent with previously reported values. Using the present method, a total amount of as few as 1.2 fmole of enzyme and 9.2 ng of substrate were injected in the capillary for the construction of a Michaelis Menten curve (seven concentrations of substrate, each concentration analyzed in triplicate), which is far smaller than the quantities required in conventional methods.
Two‐dimensional electrophoresis of human urinary proteins following concentration by dye precipitation
Authors: Marshall Thomas , Williams Katherine 
Published: May 2005

AbstractThe use of dye precipitation for concentration of proteins prior to 2‐DE has been demonstrated by application to human urine. The precipitation methods (T. Marshall and K. M. Williams, Clin. Chem. 1993, 39, 2314–2318; T. Marshall et al., Electrophoresis 1995, 16, 28–31) have been adapted for two‐dimensional electrophoresis (2‐DE) by modifying the reagent composition and adding sodium dodecyl sulphate (SDS) to the assay mixture. These modifications extend the precipitation range of the methods and overcome the problem of soluble glycoprotein‐dye complexes. The protein‐dye complex is recovered by centrifugation and dissolved in a small volume of sample denaturing buffer. The dye separates from the protein on 2‐DE (forming a sharp band at the anodal end of the isoelectric focusing gel) so that the positional coordinates of the polypeptides are unaffected by the treatment. Dye precipitation provides a simple, rapid and highly economic method for concentrating urines of low to intermediate protein content (0.02–0.50 g/L) prior to 2‐DE analysis.
Improved two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles by using immobilized pH gradients
Authors: Davidsen Nina B. 
Published: May 2005

AbstractAn improved method for two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles is presented. With the use of immobilized pH gradients in the first dimension, this method gave stable separations, with the proteins evently distributed over the entire gel surface. The number of spots in the electrophoretograms have increased fourfold compared to our earlier separations.
Genetic heterogeneity in acatalasemia
Authors: Góth László , Páy Anikó 
Published: May 2005

Abstract203 bp long products containing exon 4 and its juntions from the catalase gene were generated by polymerase chain reaction (PCR). These products were analyzed by single strand conformational polymorphism (SSCP), heteroduplex formation and nucleotide sisters, their family members and normocatalasemic controls were analyzed. Sequence analyses did not show the G to A point mutation at position 5 of intron 4. This splicing mutation characterizes the Japanese‐type of acatalasemia.
Protein electrophoresis in polyacrylamide gels with templated pores
Authors: Rill Randolph L. , Locke Bruce R. , Liu Yingjie , Dharia Jayesh , Van Winkle David 
Published: May 2005

AbstractAn approach is described for the synthesis of nanostructured hydrogels with defined size channels or pores for separations of biological macromolecules. Polyacrylamide gels (15–20% acrylamide) were cast in the presence of high concentrations (5%–28%) of hydrophilic, macromolecular cosolutes either semirigid, rod‐like polyelectrolytes (short fragments of DNA or xanthan) or spherical micelles of sodium dodecyl sulfate (SDS). Polyanionic cosolutes were then removed by a combination of diffusion and electrophoresis. These processes are expected to yield gels with ‘templated’ channels having dimensions near those of the double helical polymers (diameters ≈︁ 2–3 nm, lengths of 50–200 nm) or pores near the size of the spherical SDS micelles (≈︁ 4–5 nm). This hypothesis was tested by comparing the relative electrophoretic mobilities of proteins (3400 to 43 000 Da) complexed with SDS on templated and conventional gels. Differences in electrophoretic mobilities were observed between all templated gels and control normal gels, demonstrating that the templating process altered the polyacrylamide network. No evidence was found for phase separation of cosolutes from the polyacrylamide network during polymerization. Templated pores are expected to enhance the mobilities of molecules in a particular size range relative to smaller and larger molecules. Gels templated with DNA or xanthan (8–10% final concentration) exhibited little size selectivity, but selectivity was observed for gels templated with SDS (17–20% final concentration). The degree of selectivity of gels templated with SDS varied with polyacrylamide concentration in a manner consistent with the creation of templated pores approximately the size of SDS micelles and larger than the average pore size in a surrounding polyacrylamide network.
Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin
Authors: Bayer Edward A. , Ehrlich‐Rogozinski Sarah , Wilchek Meir 
Published: May 2005

AbstractAvidin, a positively charged egg‐white protein, aggregates extensively when mixed at ambient temperatures with anionic detergents, such as sodium dodecyl sulfate (SDS). The resultant aggregates fail to penetrate the stacking gel during polyacrylamide gel electrophoresis (PAGE). To prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a manner similar to that of streptavidin; under nondenaturing conditions (i.e., incubation of samples at room temperature), both proteins normally migrated mainly as tetramers with a tendency to form oligomers of the tetramer. When samples were boiled, both proteins migrated mainly as the monomer. The comparative stability properties of avidin and streptavidin were also examined using SDS‐PAGE by heating samples and determining the extent of dissociation of tetramers to monomers as a function of temperature. A distinctive transition temperature could be defined for individual samples. Using this assay, it was determined that, in the absence of biotin, the quaternary structure of streptavidin is more stable than that of avidin. Biotin appears to stabilize structures of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structure of the molecule using SDS‐PAGE.
Comparison of behavior of N‐substituted acrylamides and celluloses on double‐stranded DNA separations by capillary electrophoresis at 25° and 60°C
Authors: Gelfi Cecilia , Perego Marilena , Libbra Fabio , Righetti Pier Giorgio 
Published: May 2005

AbstractThe behavior of N‐substituted poly(acrylamides) and of cellulose in the separation of double‐stranded DNA by capillary zone electrophoresis (CZE) is evaluated. Contrary to previously held belief, which attributed best separations in poly(acrylamides) to small DNA fragments (typically in the 50–1000 bp size range) and in celluloses to large DNA fragments, we show that also celluloses can achieve fine sieving of short DNA sizes provided they are used at much higher concentrations than previously reported. For example, in the case of hydroxyethylcellulose (HEC), typically used at 0.2–0.8% concentrations, levels of 3% produce excellent patterns, at 25°C, in the 50–600 bp size range. If separations are conducted at 60°C, sieving is lost in most liquid polymers. However, if the concentration of HEC is raised to 6%, and that of hydroxypropylmethylcellulose (HPMC) above 1%, sieving is fully restored. Also, N‐substituted acrylamido derivatives, notably N‐acryloylaminoethoxyethanol (AAEE) and N‐acryloylaminopropanol (AAP) offer excellent performance at both 25° and 60°C, whereas plain poly(acrylamide) irreversibly collapses at high temperatures, possibly due to intrinsic instability of the amido bond under such harsh conditions. Among all the polymers investigated, an 8% solution of poly(AAP) offers the best performance and highest theoretical plates in the 25°–60°C interval. Separations at high temperatures are necessary when dealing with detection of point mutations in temperature‐programmed CZE and are preferred in DNA sequencing. Two additional advantages are evident when running DNA separations at 60°C: a marked reduction of analysis times and a linearization of the transit times of the larger (434–587 bp) DNA fragments, in all polymers tested.
A numerical study of persistence length effects on DNA conformation in sequencing electrophoresis
Authors: Guerry Emmanuel , Martin Olivier C. , Tricoire Hervé , Siebert Rainer , Valentin Luc 
Published: May 2005

AbstractWe have developed a program to evaluate the influence of DNA stiffness on molecular mobility and conformation during electrophoresis. This (currently) two‐dimensional numerical study models DNA as a chain of uniformly charged beads connected to one another by elastic segments, of finite mean size, in the presence of fixed obstacles representing gel fibers. Contrary to the standard biased reptation model (BRM), our Langevin‐type dynamics for the chain are microscopic and warrant studies of fine effects such as inner chain orientation. Using this model, we show that the introduction of a persistence length decreases the (saturated) mobility at high electric fields, providing strong evidence that the gel generates a friction force and not only a (dissipation‐free) constraint force. We also show that the persistence length leads to an increase of the chain orientation in the field direction. This suggests that DNA stiffness causes the saturation plateau value to be reached for smaller chain sizes than those predicted by the BRM model.
Simulations of the overshoot in the build‐up of orientation of long DNA during gel electropohoresis based on a distribution of oscillation times
Authors: Carlsson Christina , Larsson Anette 
Published: May 2005

AbstractThe preiodic extension‐contraction motion observed for long DNA molecules undergoing agarose gel electrophoresis in a constant field is believed to be important for the separation mechanism in pulsed field gel electrophoresis. These oscillations give rise to an overshoot and an undershoot in the ensemble orientation of DNA in the beginning of a field pulse, when the molecules oscillate coherently. After approximately one oscillation cycle, the coherence between the molecules is lost, and a constant, cycle‐averaged orientation is reached. In this paper we simulate this build‐up of the ensemble orientation of DNA by using a distribution of oscillation times (the time between two consecutive compressed conformations) determined by fluorescence microscopy for YOYO‐stained DNA. Six different orientation profiles, describing the orientation during one oscillation cycle, were used. The simulated orientation responses are compared with an orientation response measured by linear dichroism (LD) under the same experimental conditions as in the microscopy study. We found that the choice of orientation profile during the oscillation is important. Best agreement between the simulated and the experimental orientation response was obtained for an orientation profile based on a theoretical model by Schurr and Smith (Biopolymers 1990, 29, 1161–1165). The influence of the distribution of oscillation times and its standard deviation on the orientation response was also investigated. Furthermore, simulations at different field strengths and DNA sizes were performed and found to agree quite well with the experimentally obtained LD data.
Activation energy of the separation of DNA sequencing fragments in denaturing noncross‐linked polyacrylamide by capillary elecrophoresis
Authors: Fang Yu , Zhang Jian Zhong , Hou Joan Y. , Lu Hui , Dovichi Norman J. 
Published: May 2005

AbstractWe present the mobility of DNA sequencing fragments as a function of temperature in 5%T, 0%C noncross‐linked polyacrylamide and at an electric field of 150 V/cm. The mobility of the fragments follows the Arrhenius equation. The activation enegry for migration of a fragment through the polymer decreases monotonically with fragment length. The data were also analyzed in terms of the biased reptation model; the onset of biased reption with stretching occurs for longer fragments as the temperature increases. These results are quite different from those observed for separation of DNA in cross‐linked gels and represent a fundamental difference in the physics of the cross‐linked and noncross‐linked polyacrylamide.
Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers
Authors: Gelfi Cecilia , Perego Marilena , Righetti Pier Giorgio 
Published: May 2005

AbstractAnalysis of oligonucleotides (especially in regard to assessing the purity of antisense compounds) is typically performed in 18%T sieving liquid polyacrylamide, in 30% formamide and 7 M urea. Up to 600 V/cm have been reported, with transit times, for 20 to 25 oligomers, of 15–20 min. We show that the same analysis can be performed in isoelectric buffers, typically histidine (His), and in more dilute linear polyacrylamides, e.g. 10%T (at 0%C), with much reduced analysis times. A series of His concentrations has been explored, ranging from 25 to 150 mM. Best performance is obtained in 100 mM His (at pH = pI, i.e., 7.47 at 25°C), dissolved in 7 M urea, in presence of 10% sieving liquid linear polyacrylamide. Such a buffer allows delivering 800 V/cm without any loss of resolution due to Joule heating, with retention of very high resolving power down to fragments as short as tetranucleotides. Under these conditions, the analysis time for an antisense oligonucleotide containing fragments from a 10‐mer to 18‐mer is in a time window of 4–5 min. It is shown that the smallest fragment (10‐mer) migrates in the capillary at the remarkable speed of 5 cm/min.
Evaluation of capillary electrophoresis in polymer solutions with laser‐induced fluorescence detection for the automated detection of T‐cell gene rearrangements in lymphoproliferative disorders
Authors: Oda Robert P. , Wick Myra J. , Rueckert Lisa‐Marie , Lust John A. , Landers James P. 
Published: May 2005

AbstractThe rapid increase in the number of DNA‐based clinical diagnostic procedures, particularly polymerase chain reaction (PCR)‐based assays, has generated interest in analytical techniques that are less time‐consuming and labor‐intensive than traditional procedures such as polyacrylamide gel electrophoresis (PAGE). Capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) detection, which allows for rapid and sensitive detection of DNA fragments in an automated format, is well‐suited for DNA‐based clinical assays. In this study, we demonstrate the potential of CE‐LIF for the detection of PCR products from T‐cell receptor gamma (TCRγ) gene rearrangements present in monoclonal populations of lymphocytes. The presence of monoclonal populations of T‐cells is associated (but not always synonymous) with lymphocytic malignancies. Analysis of 31 patient samples, as well as sensitivity controls, demonstrated that CE‐LIF detection of monoclonal lymphocytic populations is comparable to that of PAGE‐SYBR Green I staining, and that CE‐LIF detection can be accomplished in less than 20 min. These preliminary results illustrate the potential feasibility of a CE‐based diagnostic assay for the detection of T‐cell gene rearrangements.
DNA Typing of a polymerase chain reaction amplified D1S80/amelogenin multiplex using capillary electrophoresis and a mixed entangled polymer matrix
Authors: Isenberg Alice R. , McCord Bruce R. , Koons Barbara W. , Budowle Bruce , Allen Ralph O. 
Published: May 2005

AbstractIn this study, a technique was developed to separate by capillary electrophoresis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci D1S80 and amelogenin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two different molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples samples subjected to environmental insult, and D1S80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of D1S80 and amelogenin forensic DNA samples.
Electrokinetic chromatography in suppressed electroosmotic flow environment: Use of a charged cyclodextrin for the separation of enantiomers and geometric isomers
Authors: Janini George M. , Muschik Gary M. , Issaq Haleem J. 
Published: May 2005

AbstractElectrokinetic chromatography (EKC), with negatively‐charged cyclodextrins (NCDs) added to the buffer, was conducted in polyacrylamide‐coated columns under suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of chromatography, which for brevity we term NCD‐EKC, are presented. The chiral sulfated cyclodextrin, β‐CD‐SBE (IV), used in this study is anionic over the entire pH range accessible to capillary electrophoresis, and the coated columns are stable and provide reproducible performance in the pH range 2.5–8.8. Optimum separation was obtained in the pH range where the solutes are neutral. The incorporation of an alkyl spacer between the sulfate ion and the rim of the cyclodextrin allows an unhindered approach and inclusion of neutral solutes in the cyclodextrin cavity. Solute migration time is inversely proportional to the concentration of the chiral selector. Separation (relative migration time difference) increases with decreasing chiral selector concentration and approaches a maximum, beyond which further decreases in chiral selector concentration result in broad peaks and loss of resolution. A chiral selector concentration of 1% in a 10 mM phosphate buffer produced excellent separation of amino acids and dipeptide enantiomers. In addition to being chiral selectors, cyclodextrins are also known as shape selectors. NCD‐EKC is particularly suited for the separation of positional isomers of hydrophobic solutes. The separation of aflatoxin isomers and chlorophenol congeners is presented. In the separation of chlorophenols the more hydrophobic trichlorophenols eluted first and the least hydrophobic, phenol, eluted last.
Analysis of interaction between cadmium and metallothionein isoforms by capillary zone electrophoresis
Authors: Minami Takeshi , Matsubara Hideki , O‐higashi Masayuki , Kubo Kanenobu , Okabe Nobuo , Okazaki Yuko 
Published: May 2005

AbstractThe effects of cadmium on the peak area of metallothionein (MT) protein were studied by capillary zone electrophoresis with a polyacrylamide‐coated tube at neutral pH. When cadmium was added to a commericial standard MT‐1 isoform prepared from rabbit liver, the peak area of the MT‐1 isoform decreased in a time‐ and dose‐dependent manner. The MT‐2 isoform also decreased with time, but the rate of decrease was lower than that of the MT‐1 isoform. When ethylene glycol‐bis‐(β‐aminoethyl ether)‐N,N,N′N′‐tetraacetic acid (EGTA) was added to a solution containing cadmium and MT‐1, the peak area recovered with incrasing concentration of EGTA. Zinc caused a slight decrease in the peak areas of MT‐1 and MT‐2 compared with cadmium, while addition of sodium did not decrease the areas. Furthermore, the peak areas of MT isoforms of the crude extract prepared from zinc‐treated mice also decreased with increasing concentration of cadmium. These results indicate that cadmium may change the charge of MT, which may account for the observed differences in electrophoretic behavior.