bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pulsed field agarose gel electrophoresis in the study of morphogenesis: Packaging of double‐stranded DNA in the capsids of bacteriophages
Authors: Serwer Philip , Hayes Shirley J. , Moreno Elena T. , Louie Donna , Watson Robert H. , Son Marjatta 
Published: May 2005

AbstractTo understand how comparatively simple macromolecular components become biological systems, studies are made of the morphogenesis of bacteriophages. Pulsed field agarose gel electrophoresis (PFGE) has contributed to these studies by: (i) improving the length resolution of both mature, linear, double‐stranded bacteriophage DNAs and the concatemers formed both in vivo and in vitro by the end‐to‐end joining of these mature bacteriophage DNAs, (ii) improving the resolution of circular conformers of bacteriophage DNAs, (iii) improving the resolution of linear single‐stranded bacteriophage DNAs, (iv) providing a comparatively simple technique for analyzing protein‐DNA complexes, and (v) providing a solid‐phase quantitative assay for all forms of bacteriophage DNA; solid‐phase assays are both less complex and more efficient than liquid‐phase assays such as rate zonal centrifugation. Conversely, studies of bacteriophages have contributed to PFGE the DNA standards used for determining the length of nonbacteriophage DNAs. Among the solid‐phase assays based on PFGE is an assay for excluded volume effects.
Biased sinusoidal field gel electrophoresis for the separation of large DNA
Authors: Kotaka Tadao , Adachi Shiro , Shikata Toshiyuki 
Published: May 2005

AbstractIn agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility μs versus size M relation for linear DNAs (L‐DNAs) can be divided into three regimes: Ogston regime I for small DNAs, where M dependence of μs is weak; entangled but unstretched regime II for intermediate‐size L‐DNAs (of M < 20 kbp), where μs σ M−1 so that efficient fractionation is possible; and entangled and stretched regime III for large L‐DNAs, where M dependence of μs is again weak. Although μs and the regime boundaries can be altered by adjusting the gel concentration Cgel and/or the field strength E, the features of the M dependence of μs are essentially unchanged. As to the effect of DNA topology on μs, we found that in dilute gels (Cgel < 1.0 wt%) coiled, circular DNAs (C‐DNAs) of 2–7 kbp size migrate faster than L‐DNAs of comparable size, while in concentrated gels (Cgel > 1.5 wt%) C‐DNAs migrate much slower than L‐DNAs. To facilitate separation of large DNAs in the regime III range, we proposed biased sinusoidal field gel electrophoresis (BSFGE), which utilizes a sinusoidal field of strength Es and frequency f superposed on a steady bias field of strength Eb. Striking results in BSFGE of low bias (Eb < Es) conditions were that (i) the effective mobility μ at low f (μo) is higher than that of μ∞ at high f, which is equal to the steady field value μs, and (ii) for large DNAs of M > 20 kbp the μ exhibits a minimum μp (pin‐down mobility) at a frequency fp (pin‐down frequency) specific to M, Cgel, and the field strengths in such a way that fp σ M−1 Cgel −1Eb. Esα with α changing from 0 to 2 ∼ 3 at a value of Es dependent on Eb. The μp values appear to fall on the extrapolated portion of the regime II log(μs) versus log M curve. These results are interpreted in terms of the current dynamical models of DNA gel electrophoresis and also with the results of direct observation by fluorescence microscopy on migrating T4dC DNA of 166 kbp in a steady field and under several BSFGE conditions.
Dynamics of DNA under pulsed‐field gel electrophoresis as analyzed from birefringence rise and decay
Authors: Mayer Pascal , Sturm Jean , Heitz Caroline , Weill Gilbert 
Published: May 2005

AbstractThe orientation of DNA induced by electrophoretic transport in agarose gel has been studied by optical birefringence. From its field‐free decay, it is clearly dem‐onstrated that the degree of orientation results from two processes: alignment along the electric field (stretching of the end‐to‐end vector) and elongation of the primitive path in the gel (overstretching). Separation of the two contributions allows the experimental determination of the effective charge per base pair, the gel pore size seen by the reptating molecule, the reptation time, the degree of over‐stretching and the mean relaxation time of the overstretching. Their field and DNA length dependences compare well with theoretical predictions. Similarly, the time at which overstretching presents an overshoot in the rise of the orientation follows closely the predictions of a model based on the evolution of J‐shaped conformations. The recovery of such conformations is studied by a sequence of two pulses with variable delay time. The use of directly measured or extrapolated characteristic times and fields in the design of efficient pulse schemes for pulsed‐field gel electrophoresis is emphasized.
Determination of the isoelectric points of low and high molecular mass ampholytes by capillary electrophoresis
Authors: Klepárník Karel , Šlais Karel , Boček Petr 
Published: May 2005

AbstractA methodology for a reliable and absolute determination of isoelectric points of amphoteric species by capillary zone electrophoresis is proposed. The principle of this absolute method consists in the application of an additional electroosmotic and/or hydrodynamic flow of a background electrolyte. This allows the measurement of electrophoretic mobilities of substances close to their isoelectric points where the net electric charge is very low and electrophoretic mobility tends to zero. The mobilities are measured at various pH values of the electrolyte so as to find the pH at which the substance moves through the separation column at zero mobility under the action of the additional flow only. The number of required measurements is lowered to the minimum with the help of the iterative procedure based on the Regula Falsi algorithm. The precision of the proposed method is predicted theoretically. The feasibility is demonstrated on real samples. The isoelectric points of histidine, myoglobine and ribonuclease are found from their titration curves.
Selecting DNA fragments for mutation detection by temperature gradient gel electrophoresis: Application to the p53 gene cDNA
Authors: Ke Song‐Hua , Kelly Patrick J. , Wartell Roger M. , Hunter Stephen , Varma Vijay A. 
Published: May 2005

AbstractTemperature gradient gel electrophoresis (TGGE) and related methods are widely employed to detect mutations in DNA fragments. DNA melting map calculations and GC clamps have been used to enhance the detection of mutations. While generally successful, these methods have not always revealed base changes within a DNA fragment. Previous work suggested that mutations are detected if they are in a DNA's first melting domain, and the melting domain is well separated from final strand dissociation. Two criteria from the DNA melting theory were established to determine when both of these conditions are met. The criteria involve calculating the derivative melting curve as well as the melting map of a DNA sequence. The approach was applied to the cDNA sequence of the human p53 gene. Mutations in the p53 gene are common in human cancers and are generally located in four ‘hot spot’ regions. Calculations indicated that three DNA fragments are needed to detect base substitutions in the four hot‐spot regions. Predicted melting behavior was experimentally tested with eight single base substitutions distributed among the four hot‐spot regions. All mutations tested behaved as predicted and were detected by vertical TGGE. Heteroduplex DNAs formed by melting and reannealing various ratios of wild type and mutant DNA fragments were also examined. Results indicated that point mutations can be detected by ethidium bromide staining from samples containing 10% mutant and 90% wild‐type sequences.
On the kinetics of monomer incorporation into polyacrylamide gels, as investigated by capillary zone electrophoresis
Authors: Righetti Pier Georgio , Caglio Silvia 
Published: May 2005

AbstractThe kinetics of monomer incorporation into a polyacrylamide gel have been studied in a photopolymerization system comprising 100 μM methylene blue in presence of a red‐ox system, 1 mM sodium toluenesulfinate (reducer) and 50 μM diphenyliodonium chloride (oxidizer). A precise assessment of gel point (pc) was obtained in a droplet chamber, in which argon was gently bubbled with a fused silica capillary into the reaction mixture. At pc, 50% (±3) acrylamide was incorporated into the matrix, vs. 80% (±4) N,N′‐methylenebisacrylamide. This incorporation level remained the same when polymerized in the 2–36°C temperature range. Incorporation continued almost linearly for acrylamide up to 80% conversion. The reaction was continued up to 55 min (at 2°C), at which point bisacrylamide had been essentially consumed (>99.5% incorporation) and acrylamide had reacted (95 %). At 2°C, after gelation, the gel became progressively turbid (the Tyndall effect plateauing at 50 min), but it remained fully transparent if, at the gel point, reaction was continued at 50°C. The consumption of the pendant double bonds of Bis followed the progression of turbidity. It is concluded that, by gelation at 2°C, the nascent chains form clusters held together by hydrogen bonds (melting point at 28°C); such clusters are subsequently “Frozen” in the three‐dimensional space as the pendant double bonds in the chains react progressively. Such turbid matrices are more porous and less elastic than when the gel is polymerized at 50°C. This process is similar to the “lateral aggregation” occurring when gels are formed in presence of a polymer in solution (e.g. 10 KDa polyethylene glycol; Righetti et al., Electrophoresis 1992, 13, 587–594).
Pulsed‐field acceleration: The electrophoretic behavior of large spherical particles in agarose gels
Authors: To Kwong‐Yuk , Boyde Tom R. C. 
Published: May 2005

AbstractIn agarose gel electrophoresis, periodically inverting or interrupting the applied field may greatly accelerate the migration of polystyrene microspheres, in a manner varying with pulse times, and the observed zones are made sharper. The particles concerned are just large enough that under constant field they appear not to enter the gel at all or to migrate very slowly: and merely lowering the applied field may also enhance their electrophoretic migration, though to a lesser extent than with field pulsing. These effects may be accounted for by gel mesh flexibility which, varying with the nature of the migrating species, may either help or hinder migration.
Clinical and forensic applications of capillary electrophoresis
Authors: Thormann Wolfgang , Molteni Sarah , Caslavska Jitka , Schmutz André 
Published: May 2005

AbstractThis survey is aimed at giving the readers a short overview of the present state of the art of clinical and forensic applications of capillary electrophoresis. First, the principles associated with electrokinetic capillary separations and instrumentation, sample preparation and solute quantitation are briefly discussed. This is followed by chapters describing the determination of endogenous and exogenous compounds in body fluids and tissue extracts. Finally, a survey of major achievements including reference to fully developed electrokinetic capillary assays is provided. The paper concludes with a brief outlook.
Determination of 2—5A synthetase and 2—5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: Effects of cytokines and comparison with radioenzymatic methods
Authors: Bruchelt Gernot , Büdenbender Manfred , Schmidt Karl‐Heinz , Birk Anette , Treuner Jörn , Niethammer Dietrich 
Published: May 2005

Abstract2—5A Synthetase and 2—5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2—5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon‐related cytokines (tumor necrosis factor‐alpha and interleukine‐2) on 2—5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2—5A synthetase in these cells. 2—5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2′p5′A2′p5′A (trimeric form of 2—5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus‐32 labeled 2—5A cores was registrated by radioenzymatic assay. Activity of 2—5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2—5A synthetase as well as of 2—5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2′p5′A2′p5′A, respectively.
Separation of enantiomers of N‐acetylcysteine by capillary electrophoresis after derivatization by o‐phthaldialdehyde
Authors: Dette Christoph , Wätzig Hermann 
Published: May 2005

AbstractThe optical purity of N‐acetyl‐L‐cysteine was tested by capillary electrophoresis. The D‐enantiomer can be found down to a ratio of 0.4:99.6 within 4 min without the use of the D‐form or the racemate as a standard. The efficiency was optimized during method development up to a theoretical plate number, N, of several hundred thousand. The migration order and the separation mechanism was explained. The assumed structure of the formed isoindole S‐[2‐(1‐carboxy‐2‐methylpropyl)isoindole‐1‐yl]‐N‐acetylcysteine diastereomers was confirmed and its stability was examined.
Introduction of migration indices for identification: Chiral separation of some β‐blockers by using cyclodextrins in micellar electrokinetic capillary chromatography
Authors: Sirén Heli , Jumppanen Juho H. , Manninen Kirsi , Riekkola Marja‐Liisa 
Published: May 2005

AbstractBecause of the different physiological impact that stereoisomers may have, it is often vital to separate these forms from one another. Because of their structural similarity, the separation is usually difficult to achieve and zones may elute very close to each other. This is a particular problem in capillary electrophoresis, where the repeatabilty of absolute migration times is fairly poor, mainly due to the irreproducibility of the electroosmotic flow. The separation is usually repeatable, however, and when the disturbing effects are eliminated by using a migration index system incorporating two marker compounds the identification of the enantiomers becomes extremely good. Relative standard deviation (RSD) values less than 0.1% for the migration index of each enantiomer were obtained in both intra‐day and day‐to‐day (6 days) studies. The best separation was achieved with the electrolyte solution made of 40 mM borate, 32 mM sodium dodecyl sulfate (SDS), 12 mM β‐cyclodextrin (β‐CD), and 6 mM α‐cyclodextrin (α‐CD) at pH 9.3.
Separations of derivatized amino acid enantiomers by cyclodextrin‐modified capillary electrophoresis: Mechanistic and molecular modeling studies
Authors: Copper Christine L. , Davis Joe B. , Cole Roderic O. , Sepaniak Michael J. 
Published: May 2005

AbstractThe enantiomers of 5‐dimethylamino‐1‐naphthalene sulfonyl (DNS)‐deriva‐tives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio‐selective running buffer additives. A previously described model for retention and chiral recognition in CD‐modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid‐CD complexation constants for enantiomer pairs. In this work, the D‐enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co‐elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS‐amino acid enantiomer‐CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer‐CD interaction energies is shown to provide reasonable correlation with separation performance.
Enhanced chiral separation of dansylated amino acids with cyclodextrin‐dextran polymer network by capillary electrophoresis
Authors: Sun Peng , Barker Geoffrey E. , Mariano Greg J. , Hartwick Richard A. 
Published: May 2005

AbstractA method for the chiral separation of dansyl‐DL‐amino acid mixtures using a dextran (Mr 2 000 000) polymer network containing β‐cyclodextrin (β‐CD) in HPCE was developed. Mixtures of up to seven amino acids could be baseline resolved by this method under neutral pH conditions. Resolution was dependent on the dextran concentration in the polymer network. Temperature effects on the chiral separation were studied. Optimal efficiency and resolution of DL‐enantiomeric pairs of amino acid samples were obtained at 25°C. The resolution of different amino acids used in this work decreased with an increase in temperature.
Pharmaceutical and biomedical applications of capillary electrophoresis/electrochemistry
Authors: Lunte Susan M. , O'Shea Thomas J. 
Published: May 2005

AbstractThe use of capillary electrophoresis/electrochemistry (CEEC) for the analysis of microdialysis samples obtained for pharmacokinetic and neurochemical studies is described, as well as the development of new types of electrodes and waveforms which increase the selectivity of this technique for specific classes of analytes. CEEC with a carbon fiber electrode was employed for the analysis of microdialysis samples. Microdialysis is an in vivo sampling technique that yields very small samples for analysis (less than 1 μL). Therefore, capillary electrophoresis, with its small volume requirements, is an excellent choice for the analytical method. CEEC was used to study the pharmacokinetics of L‐dopa and the release of aspartate and glutamate following a high K+ infusion in the brain. Several modified electrodes which increase the applicability of CEEC in pharmaceutical and biomedical analysis are described. One of these is a gold/mercury electrode which is highly selective for thiols and was used for the determination of glutathione in a rat brain. An alternative method for the detection of thiols employed a chemically modified electrode containing cobalt phthalocyanine. In this case, an electrocatalyst reduces the overpotential of thiols at the carbon electrode and makes it possible to detect them at a much lower and more selective oxidation potential. This electrode was used for the detection of cysteine in urine. The development of pulsed amperometric detection for capillary electrophoresis is also described and is demonstrated by the detection of glucose in blood. Lastly, a method for the detection of peptides based on the formation of a copper complexs and detection at a carbon fiber electrode is discussed.
Multifunctional apparatus for electrokinetic processing of proteins
Authors: Horvath Z. Stephen , Corthals Garry L. , Wrigley Colin W. , Margolis Joel 
Published: May 2005

AbstractNew equipment (the “Gradiflow TM”) has been designed and constructed to provide efficient large‐scale preparative fractionation of macromolecules, based on charge and/or size differences, as well as the concentration of macromolecules and electrodialysis. Examples of its capability are the separation of a mixture of haemoglobin (50 mg) from bovine serum albumin (50 mg) within 15 min (based on charge differences at pH 6.8), the purification of phycoerythrin from a crude extract on the basis of size, and the fractionation of serum proteins into two discrete size classes.
Is gravity on our way? The case of polyacrylamide gel polymerization
Authors: Righetti Pier Giorgio , Bossi Alessandra , Giglio Marzio , Vailati Alberto , Lyubimova Tatyana , Briskman Vladimir A. 
Published: May 2005

AbstractPotential gravity‐induced deformations of polyacrylamide matrices during gelling were investigated in two different initiator systems based on (i) photopolymerization with 100 μM methylene blue, 1 mM sodium toluene sulfinate (reducer) and 50 μM diphenyliodonium chloride (oxidizer) (photopolymerization) and (ii) chemical polymerization, utilizing the standard persulfate N,N,N,N‐tetramethylethylenediamine. In both systems, it is seen that convective flows are imprinted in the final gel structure above a critical gelling layer thickness, set at ca. 3 mm. In both systems, progressive increments of the solution density, from normodense (density = 1.0) up to isodense with the growing polymer chains (density = 1.3) do not inhibit the appearance of strong convective flows. However, gel inhomogeneities are completely abolished even in 10 mm gelling layers if polymerization is performed in presence of density gradients, notably of sucrose, from 0 to 20%, 0 to 40% and 0 to 60%. Even the shallower gradient (0–20% sucrose) is able to completely abolish convective flows in persulfate‐driven polymerization. It is hypothesized that such disturbances are not created by sedimentation of the growing polymer chains in the gravitational field, but are produced by the reaction exothermality, which produces strong buoyancy‐driven flows. It is additionally demonstrated that persulfate polymerization is sensitive to oxygen absorbed from the top liquid layers, which should be carefully protected by an overlay of organic solvent. Methylene blue‐induced polymerization appears to offer a series of unique advantages over chemical initiation with persulfate.
Analysis of photoreceptor proteins of microorganisms by gradient gel electrophoresis and other biochemical separation methods
Authors: Häder Donat‐P. , Lebert Michael 
Published: May 2005

AbstractPhotoreceptor proteins for photoorientation in microorganisms are usually membrane bound and can be isolated by standard biochemical methods. Three examples are shown: the flagellates Euglena gracilis, Peridinium gatunense and the slime mold Dictyostelium discoideum. The photoreceptor of Euglena is attached to the basis of the flagellum and is composed of at least four chromoproteins which can be separated by gradient sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) fast protein liquid chromatography (FPLC) and isoelectric focusing (IEF); it contains pterins and a flavin as chromophoric groups. The photoreceptor of Peridinium absorbs in the red wavelength band. Though not yet identified in detail, multiple receptors are probably involved, as indicated by fluorescence spectroscopy. Dictyostelium shows positive and negative phototaxis in its amoebal form and exclusively positive phototaxis in its pseudoplasmodial form. It is still open to discussion whether the two stages use separate photoreceptors. From amoebae two photoreceptor pigments have been isolated, showing an absorption which resembles the action spectrum, one membrane bound with a molecular mass of 45 kDa and one cytoplasmic fraction with a molecular mass of 27 kDa.