bluuebunny/crossref_metadata_2025_split huggingface dataset:
Computerized analysis of double‐label autoradiographs: A screening method for 1,25‐dihydroxyvitamin D3 induced changes in intestinal protein synthesis
Authors: Kendrick Nancy C. , Eakle Kurt , Deluca Hector F. 
Published: May 2005

AbstractThe McIDAS computer system (Man‐computer Interactive Data Access System) at the University of Wisconsin was used to analyze double‐label autoradiographs of two‐dimensional (2‐D) electrophoresis gels. The 4 samples used to generate the 2‐D gels were obtained from 8 vitamin D‐deficient rat intestines 25 h after injection with 1,25‐dhydroxyvitamin D3 (1,25‐(OH)2 D3) or vehicle. To perform the analysis, the patterns on Kodak XAR5 (XR) film, which detects 14C + 3H by fluorography at ‐75 °C, and on Kodak NS‐2T (NS) film, which detects 14C only by direct autoradiography at room temperature, were converted to digital images. The McIDAS system was used to multiply the NS image by a normalization factor which matched it to the XR image with respect to exposure. After background subtraction from both images, the XR image was divided by the aligned normalized NS image; the resultant ratio image was multiplied by an enhancement factor and colored according to pixel brightness. Polypeptides with a high or low ratio relative to the average ratio stood out clearly in contrasting colors on the ratio image and could be detected by visual inspection. The actual XR/NS density ratios of these highlighted spots could then be quantitated by spot integration. To test the method, XR/NS density ratios were measured for 34 spots on each of the 4 films, sets and the spot ratios compared to the spot appearances on the ratio image. With the exception of a few false positives, agreement was good. Several polypeptides were found to be increased by 25–50 % relative to the average ratio while one polypeptide of molecular weight 50 000, approximate pI 5.5, was increased an average of 240 %.
Formation of natural pH gradients in sequential moving boundary systems with solvent counterions. I. Theory
Authors: Hjelmeland L. M. , Chrambach A. 
Published: May 2005

AbstractThe moving boundary theory has been applied to the special case in which the proton and hydroxyl ion are the sole counterions. Moving boundaries between two monovalent weak acids, two biprotic acidic ampholytes, two weak bases and two biprotic basic ampholytes were considered. This application of the theory leads to constituent concentration relationships, values for pH and conductance of each phase and step pH gradients when sequential moving boundaries are considered. In addition, rigorous conditions for achieving the steady‐state in such systems are also presented. Application of the theory allows for the prediction of the formation and displacement of pH gradients. In contrast to the classical theory of pH gradients formed by ampholytes, the establishment of inverse pH gradients and the electrophoretic displacement of natural pH gradients is clearly accounted for.
The distribution of and the biochemical and serological relationships between the I/i and ABH blood group antigens of the human erythrocyte membrane as determined by immunoelectrophoretic techniques
Authors: Oppenheim Joel D. , Nachbar Martin S. , Blank Margild 
Published: May 2005

AbstractMacromolecules isolated from human erythrocyte membranes which expressed I/i and ABH blood group activities were analyzed by quantitative immunoelectrophoretic techniques in order to determine the distribution of and the relationship between these two blood group systems. To help in these studies a rocket immunoelectrophoretic assay, with a maximum sensitivity of 10 ng, was developed which could detect and quantitate erythrocyte macromolecules which expressed I/i and A and B blood group determinants. In addition, a modification of the tandem crossed immunoelectrophoresis technique was also used to elucidate the structural relationship between determinants of these systems on one of the isolated macromolecular species. Serological and other immunological data are presented which throughly support the immunoelectrophoretic results. From these studies we have concluded that I and most likely i antigenic determinants are primarily expressed on poly(glycosyl)ceramides (i. e. macroglycolipids) and possibly to a lesser extent on Band 3. I active poly(glycosyl)ceramide could be isolated in equal quantity from any adult ABH type erythrocyte; i activity was found in similar quantity in the same fraction isolated from one i adult erythrocyte population. Besides expressing I/i activity the various poly(glycosyl)ceramide fractions also exhibited A, B or H activity as well, dependent upon the ABH cell type from which they were isolated. Unlike the equal distribution of I, however, ABH activity was type‐specific in that the poly(glycosyl)ceramide isolated from type A (either A or Ai), B or 0 cells could react only with anti‐A, anti‐B, or Ulex anti‐H lectin, respectively. Using the modified tandem crossed immunoelectrophoresis (CIE) technique, it was established that the same glycolipid molecules which carry I blood group activity express the A and B determinants as well. Band 3 material isolated from 0 erythrocytes exhibited I, i and H activities. From CIE analysis the I activity of Band 3 appeared to reside in a subpopulation of this material.
Detection of the major non‐serum derived proteins of body fluids by immunodeletion and two‐dimensional gel electrophoresis
Authors: Dermer Gerald B. , Edwards Jesse J. 
Published: May 2005

AbstractCommercial antisera to human serum were coupled to Sepharose 4B or Staphylococcus aureus bacteria and used as immunoadsorbents to separate the serum‐derived proteins in several types of body fluids from the fluid‐specific, non‐serum derived proteins. Two‐dimensional gel analysis of fluids after incubation with one or the other immunoadsorbent produced similar results. Cerebrospinal, prostatic and amniotic fluid each exhibited a characteristic array of probable fluid‐specific protein families which were not detected in serum. Data from model experiments suggested that Sepharose 4B was a somewhat better matrix material than Staphylococcus aureus cells. Pretreatment of body fluids with anti‐human serum immunoadsorbents should facilitate the detection of fluid‐specific protein markers of disease in two‐dimensional gels.
Agarose gel electrophoresis of bacteriophages and related particles. III. Dependence of gel sieving on the agarose preparation
Authors: Serwer Philip , Allen Jerry L. , Hayes Shirley J. 
Published: May 2005

AbstractThe sieving of different preparations of agarose has been measured during agarose gel electrophoresis, using as samples a spherical bacteriophage and two spherical bacteriophage‐related particles with radii between 13.3 and 41.9 nm. Semilogarithmic plots of electrophoretic mobility as a function of agarose perentage (A) were linear for A ⩽ 0.85, but became progressively nonlinear as A increased above 0.85. The regions of linearity in these plots were used to measure gel sieving and to determine an effective gel fibre radius. The sieving of the different preparations increased in the order HGT[P] < ME < Seaplaque (hydroxyethylated) Isogel (additive containing) < SeaPrep 15/45 (hydroxyethylated) (trade names of preparations from Marine Colloids). However, the effective gel fibre radius did not differ significantly from 25 nm among HGT[P], ME, SeaPlaque and SeaPrep 15/45 agarose; for Isogel, this radius was 20 nm. These data have been interpreted to indicate that the comparatively high sieving of SeaPlaque, SeaPrep 15/45, and to some extent Isogel agarose was caused by a comparatively low agarose mass per fibre volume and high total fibre length per gel volume. For at least some particles smaller than those used here, it is likely that migration through the gel fibres detected here occurs during agarose gel electrophoresis.
Isoelectric focusing in immobilized pH gradients in presence of urea and neutral detergents
Authors: Gianazza Elisabetta , Artoni Gianfranco , Righetti Pier Giorgio 
Published: May 2005

AbstractThe pK's of Immobilines, the reactive acids and bases used for establishing the pH in polyacrylamide gel matrices for isoelectric focusing with immoblized pH gradients, were measured in presence of urea (up to 8 M) and a neutral detergent'(2 % Nonidet P‐40). The temperature dependence of the pK's in 8 M urea was also investigated. ΔpK's due to the addition of urea were found to be higher for the acidic than for the basic Immobilines. Formulations are given to obtain 4 and 5 pH unit wide gradients with pre‐fixed extremes and linear in the presence of 8M urea. Such gels require, as pretreatment before the run is started, either an electrophoretic elution, by applying the voltage perpendicular to the direction of the pH gradient, or extensive washing in water, followed by drying and re‐equilibration in urea solution.
Polymorphism of soluble rat brain guanylate cyclase is produced during isoelectric focusing
Authors: Basset Paul , Zwiller Jean , Revel Marie‐Odile , Vincendon Guy 
Published: May 2005

AbstractSoluble guanylate cyclase purified from rat brain exhibits about 30 different components between pH 5.3 and 6.5 on polyacrylamide gel isoelectric focusing (IEF) Specific immunodetection after IEF of guanylate cyclase from fresh rat brain supernatant allows the detection of a complex profile, indicating that the complex pattern observed with purified enzyme is not related to sample aging. In contrast, purified enzyme gives only one major band on electrophoresis in presence or absence of sodium dodecyl sulfate and a single line of immunoprecipitation was obtained on crossed immunoelectrofocusing. The IEF pattern exhibited by guanylate cyclase is dependent on the sample concentration, the pH range of sample application, the carrier ampholytes used and on the presence of urea in the gel. Fractionation tests, performed with partially purified enzyme or with purified enzyme, indicate that this complex pattern is produced during IEF and does not correspond to an inherent heterogeneity of soluble rat brain guanylate cyclase. The distribution of carrier ampholytes used to form the pH gradient and that of guanylate cyclase bands was found to be very similar at the steady‐state of IEF, suggesting that interaction between carrier ampholytes and rat brain soluble guanylate cyclase can be involved in the generation of the complex IEF pattern.
Artefacts associated with Nonidet P‐40 upon sodium dodecyl sulphate polyacrylamide gel electrophoresis of protein
Authors: Marshall Thomas 
Published: May 2005

AbstractOne‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of protein in the presence of Nonidet P–40 (NP–40) induces vertical streaking which breaks horizontal bands of protein to produce a “multiple spot” effect. The same effect can be induced upon high resolution two‐dimensional electrophoresis if the NP–40 concentration of the first‐dimension isoelectric focusing (IEF) gels is increased to 4 % w/v and the gels electrophoresed (SDS‐PAGE) without SDS‐equilibration. The relevance of this phenomenon as regards interpretation of two‐dimensional electrophoretic patterns is discussed.
Electrophoretic demonstration of interactions between group specific component (vitamin D binding protein), actin, and 25‐hydroxycholecalciferol
Authors: Emerson David L. , Galbraith Robert M. , Arnaud Philippe 
Published: May 2005

AbstractGroup specific component (Gc) is known to interact with both vitamin D3 metabolites and actin. The present study was undertaken to determine if analytical isoelectric focusing could form the basis of a simple and reliable method for discriminating native Gc and complexes with 25‐hydroxycholecalciferol (25(OH)D3) from those formed with actin and with both ligands. Increasing amounts of G‐actin and/or 25(OH)D3 were added to purified Gc of both Gc1 and Gc2 phenotypes. Actin and 25(OH)D3 interacted independently and simultaneously with native Gc, giving rise to three different complexes of increasing acidity: Gc‐25(OH)D3, Gc‐actin, and actin‐Gc‐25(OH)D3, which were clearly resolved from the native Gc protein and from each other. In addition, the inherent differences in microheterogeneity and polymorphism between the two major phenotypes examined (Gc1 and Gc2) were also retained in their respective complexes. Similar results were obtained upon addition of 25(OH)D3 or actin to whole human serum, the bands corresponding to native or complexed Gc being recognized in this case by print immunofixation. These results indicate that complexes formed between Gc protein and both 25(OH)D3 and actin can be clearly detected and resolved by electrophoretic procedures.
Analysis of normal human aqueous humour proteins by two‐dimensional gel electrophoresis
Authors: Segers Jeanine , Rabaey Marcel , Van Oye Rafaël 
Published: May 2005

AbstractHigh resolution two‐dimensional electrophoresis combined with a sensitive silver stain has been used to study the proteins of normal human aqueous humour. The protein patterns of small, unconcentrated, individual samples and of pooled and concentrated aqueous humour samples were compared with plasma or serum from the same individual or with a normal serum. The protein maps of aqueous humour and serum showed great similarity. More than 30 groups of spots were visualized in aqueous humour after silver staining and 18 of them have been tentatively identified. Three polypeptide groups were observed which were not found in plasma or serum. The different clusters, which appeared in the region of high (> 90 000), medium (50 000–45 000) and low (25 000–15 000) molecular weight polypeptides, remain unidentified. The most abundant cluster represents a molecular weight of approximately 48 000.
Computer‐aided standardization of two‐dimensional electrophoresis: Improvement of biophysical description of proteins in relative versus absolute coordinate systems
Authors: Fosslien Egil , Prasad Rameshwar , Stastny Jaroslav 
Published: May 2005

AbstractProtein migration distances on two‐dimensional (2‐D) gels were measured in mm (absolute) and in relative internal standard units using creatine kinase carbamylation trains (x‐axis) as relative charge (CK‐Cr) scale and rat heart extract lines as relative molecular weight (Mr) scale (y‐axis). Variation in absolute protein location generally increases with increasing migration distance in both axes with 6–12 mm and 5–11 mm for selected spots in the x‐ and y‐axis respectively; variation in relative migration is significantly less, ranging from 0.9–2.5 mm and 3–4 mm in the x‐ and y‐axis respectively; data are from seven separations run in the same batch, measurements obtained from 1.6 × magnified projections of photographed gels. A computerized plotter was used to generate scaled relative charge ‐ relative molecular weight (Cr‐Mr) plots for each separation based upon absolute positions of internal protein standard spots (first dimension) and lines (second dimension). The results support the use of computer‐delineated‐relative coordinate systems (Cr‐Mr plots) for biophysical description of proteins separated by 2‐D electrophoresis.
Polyacrylamide gel electrophoresis of small peptides
Authors: West Michael H. P. , Wu Roy S. , Bonner William M. 
Published: May 2005

AbstractTwo polyacrylamide gel electrophoretic systems for the separation of small peptides are described. In both systems the samples are concentrated by a buffer discontinuity, and the peptides separated according to size and charge in 40–50 % polyacrylamide resolving gels. The acidic gel has an operative pH between 3.0 and 3.1 while the alkaline gel has an operative pH between 9.1 and 9.2. The lower limit of resolution is about 100 daltons for a peptide with one positive charge in the acidic system. The radioactive peptides are detected by autoradiography. Peptides with a free amino group can be fixed in these gels with formaldehyde, so that fluorographic procedures can also be used for increased sensitivity. These techniques enable precise comparison of non‐derivatized peptides from complete protein fingerprints and offer a simple one‐dimensional analytical method for protein comparison, as well as a general method for separating and analyzing small peptides.
The electric field strength across isoelectric focusing gel slabs determined by an accurate and convenient method
Authors: Murel Andrei , Vilde Svetlana , Kongas Ago , Kirret Oskar 
Published: May 2005

AbstractA simple and accurate method for the monitoring of the voltage distribution across horizontal slab gels in isoelectric focusing is described. Voltage is measured by passing a single Pt wire electrode (connected to an external voltmeter and to one of the electrodes through the power supply) through holes in the apparatus lid, bored at 5 mm distance from one another across the migration path. Conductances along the migration path calculated from direct voltage measurements are by one order of magnitude lower than those measured by conductimetry on gel slice eluates. Segmental resistances calculated from the measured voltages across adjacent holes in the lid during isoelectric focusing (IEF) add up to the total resistances across the gel obtained from the voltage and current readings on the power supply. In confirmation of previous reports, segmental resistance in IEF was shown to increase dramatically with time, with maxima shifting progressively from pH 4 to 5 in a representative pH3‐10 gradient. Joule heat production in IEF decreases with increasing molecular weight of the carrier ampholytes.
Electrophoretic mobility and isoelectric point of purified brush border membrane vesicles
Authors: Ohsawa Kazuaki , Ohshima Hiroyuki 
Published: May 2005

AbstractThe electrophoretic mobility of brush border membrane vesicles purified from the rabbit ileum by chromatography on porous glass beads has been measured with a Laser‐Zee microelectrophoresis instrument. The value obtained was measured in a solution containing 0.1 M NaCl, 10 mM HEPES‐Tris and 50 mM mannitol at pH 7.37 and 20 °C was ‐1.41 μm s−1 V−1 cm. The mobility depended on both pH and ionic strength of the solution. The isoelectric point was observed to be at pH 4.25. The brush border membrane vesicle surface was found to be highly negatively charged: there are 1000–2000 negative charges on each vesicle at ionic strength 0.1. Treatments with cation had an effect on mobility and gave a reversal of surface charge.
Electrophoretic characterization of human monocytes and lymphocytes before and after stimulation with concanavalin A
Authors: Bauer Johann , Hannig Kurt 
Published: May 2005

AbstractHuman mononuclear cells were split into platelets, monocytes and small lymphocytes by countercurrent centrifugal elutriation (CCE). Determination of the electrophoretic mobilities (EM) of each population revealed that total small lymphocytes, i. e. B, T and null cells, have an equal EM at 1.09 × 10−4 (cm2 V−1 s−1), whereas monocytes showed a symmetric distribution curve with a peak at 0.95 × 10−4 (cm2 V−1 s−1). Thus the cathodal shoulder of the EM distribution curve of total mononuclear cells is caused only by monocytes. Mononuclear cells were stimulated by concanavalin A. Growing and resting lymphocytes were isolated at different times by CCE and analysed by cell electrophoresis. In contrast to murine T cells, human Tlymphocytes did not change their EM within 3 days after mitogenic transformation.
The effect of phytohemagglutinin on the electrophoretic mobility of purple membrane particles
Authors: Kantcheva Maria R. , Popdimitrova Nezabravka G. , Stoylov Stoyl P. , Kovatchev Dimiter E. 
Published: May 2005

AbstractPhytohemagglutinin (PHA)‐the lectin extracted from Phaseolus vulgaris‐is found to induce electrophoretic mobility changes of purple membrane particles. The effect is dose‐dependent. The ionic strength of the suspending medium modifies the reduction of the membrane surface charge density.
An improved electrophoretic transfer (electroblotting) apparatus
Authors: Shuttleworty Andrew D. 
Published: May 2005

AbstractAn improved apparatus for the electrophoretic transfer of proteins from gels to immobilising matrices is described. This device overcomes problems common to previous designs such as entrapment of gas bubbles within the gel sandwich during its assembly and from electrolytic gas generation during transfer. The apparatus has some novel features which facilitate its ease of use and enable several gels to be blotted at one time.
Determination of thiodiacetic acid in urine of people exposed to vinyl chloride by analytical capillary isotachophoresis
Authors: Křiváková Ludmila , Samcová Eva , Boček Petr 
Published: May 2005

AbstractA method is introduced for the determination of thiodiacetic acid in urine by analytical capillary isotachophoresis with colunm coupling. Thiodiacetic acid is one of the final metabolites of carcinogenic vinyl chloride and appears at increased levels in the urine of people exposed to vinyl chloride vapours. The method enables the direct analysis of samples of 10 μl of urine without any pretreatment in about 45 min. The lowest detectable concentration of thiodiacetic acid was about 6 × 10−6 mol/liter and the reproducibility of the analyses in the range of 2 ‐ 15 × 10−5 mol. per liter was about 3 rel. %.
Sodium salicylate for fluorographical detection of immunoprecipitated proteins in agarose gels
Authors: Heegaard Niels H. H. , Hebsgaard Kim P. , Bjerrum Ole J. 
Published: May 2005

AbstractFilm detection of compounds labeled with the weak beta‐emitters 3H and 14C is demonstrated for electroimmunoprecipitated proteins in agarose gels by means of the water‐soluble fluorophor sodium salicylate. The degree of enhancement of film detection of tritium and carbon‐14 with 0.7 M sodium salicylate paralleled fluorography with diphenyloxazole on polyacrylamide gels. The method was optimized and the sensitivity, expressed as radioactivity per cm immunoprecipitate, was 0.9 nCi/cm (2000 dpm/cm) in 24 h for 3H and 0.4 nCi/cm (830 dpm/cm) in 18 h for 14C. The procedure was found superior to the commercially available enhancement solutions ‘EnHance’ and ‘Amplify’. Salicylate fluorography on Kodak X‐Omat L and AR‐5 films was about 30 and 60 times more sensitive than Ultrofilm 3H. With 14C‐labeled anti‐antibodies rocket electroimmunoprecipitation corresponding to about 50–100 pg protein could be visualized in 7 days. Conclusively, the method is rapid (soaking and drying require about 30 min), sensitive, reproducible, cheap, and advantageous if the immunoplates are to be stained for protein after fluorography.
The behavior of serum albumin upon isoelectric focusing on immobilized pH gradients
Authors: Gianazza Elisabetta , Frigerio Adele , Astrua‐Testori Silvia , Righetti Pier Giorgio 
Published: May 2005

AbstractIsoelectric focusing on immobilized pH gradients dissociates most complexes between fatty acids and serum albumin from whole plasma. Most albumin thus migrates to a pI of 5.8, very close to the theoretical value of 6.0 as calculated with the Linderstrøm‐Lang equation. The phenomenon was elucidated through saturation experiments and time‐course monitoring of the protein pattern upon focusing.
Comparison of detection limits for various nitrocellulose binding immunoassays using β2‐microglobulin as a model antigen
Authors: Vissing Henrik , Madsen Ole D. 
Published: May 2005

AbstractThe detection limits for antigen in immunodot blots on nitrocellulose paper using β2‐microglobulin as a model antigen were determined. the binding of a mouse monoclonal antibody against β2‐microglobulin to various concetrations of purified human β2‐microglobulin was tested with six different methods. The most sensitive assay allowed the detection of about 40 pg/mm2 protein.