bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of cationic buffer additives on the capillary electrophoretic separation of serum transferrin from different species
Authors: Oda Robert P. , Landers James P. 
Published: May 2005

AbstractThe presence of specific transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human transferrin has become increasingly important. As a means of evaluating the potential for developing a rapid capillary electrophoresis‐based assay for the analysis of carbohydrate‐deficient transferrins (CDTs), the capillary zone electrophoretic (CZE) analysis of Tfs from several species was evaluated using uncoated capillaries and a separation augmented with cationic additives. With bovine Tf, five peaks (representing different sialylated forms) were partially resolved in borate and baseline‐resolved when 1,4‐diaminobutane was added to the buffer. These same conditions were found to be inadequate for the resolution of the sialo‐forms from other species. Some success was achieved using α,γ‐bis‐quaternary ammonium alkanes instead of the 1,4‐diaminobutane and optimizing the pH for each of the species Tfs. Human Tf was found to be resolved in an uncoated capillary equilibrated with a borate buffer containing millimolar concentrations of decamethonium bromide as a buffer additive. Under these conditions, resolution of the various sialoforms from the iron‐saturated Tf was possible and the glycoforms were found to migrate differently than their iron‐depleted counterparts. Despite the resolution achievable under these conditions, the lengthy analysis time is incompatible with the requirements for a clinical CZE‐based assay.
Dynamics of DNA molecules in agarose gel under sinusoidal electric fields
Authors: Starchev Konstantin , Sturm Jean , Weill Gilbert 
Published: May 2005

AbstractThe dynamics of DNA molecules in agarose gel has been studied by measuring the birefringence in sine wave electric fields. It was found that the birefringence is the sum of a direct current (dc) component and several harmonics of the field frequency. A detailed study of these components was performed. It was found that the dynamics of DNA molecules under these conditions can be explained by a W conformation which is not detected under dc electric fields. The variation of the field frequency gives several relaxation modes in the birefringence signal, reflecting the known dynamic behavior of DNA molecules found in the reptation with elongation of the tube length regime.
Sodium chloride in preparative free‐flow cell electrophoresis: Recent developments
Authors: Bauer Johann , Weber Gerhard 
Published: May 2005

AbstractThree buffer systems for free‐flow electrophoresis have been designed, which proved useful for performing cell electrophoresis in the presence of 50 mM NaCl. Each system consists of one central cell suspension buffer with 50 mM NaCl, two margin buffers, and two electrode buffers. With the aid of a bromophenol blue/maxilon blue accumulation test the various buffers were adjusted to ensure a laminar flow and remain unchanged on their way through an electrophoresis chamber.
Immunoglobulin G galactosylation deficiency determined by isoelectric focusing and lectin affinoblotting in differential diagnosis of rheumatoid arthritis
Authors: Kötz Kerstin , Hänsler Marion , Sauer Heidemarie , Kaltenhäuser Sylke , Häntzschel Holm 
Published: May 2005

AbstractIgG galactosylation deficiency in patients with rheumatoid arthritis (RA) can be detected by isoelectric focusing (IEF)/lectin affinoblotting. We analyzed IgG glycosylation in patients with early arthritis (n = 50) and healthy controls in order to determine the clinical value of this parameter in differential diagnosis of RA. A significant correlation between the IgG galactosylation defect at disease onset and the diagnosis of RA during the follow‐up was observed. Involvement of other clinical parameters (erythrocyte sedimentation rate, C‐reactive protein, rheumatoid factor) did not improve the predictive value of IgG galactosylation changes.
Phenotyping of esterase D and acid phosphatase by rapid isoeletric focusing on PhastSystem
Authors: Yaojun Guo , Lili Huang , Xuegang Yao , Zhongyi Yu 
Published: May 2005

AbstractA rapid isoelectric focusing method on PhastSystem was applied to investigate the distribution of esterase D (ESD) and acid phosphatase (ACP) phenotypes in 152 and 112 unrelated healthy donors, respectively, from the Han population in Beijing. Laboratory‐made gels with a pH range of 5.0–7.0 and 5.0–8.0 were used for analysis of ESD and ACP, respectively, with 1 μL sample loading. The running time was 30–40 min. Gene frequencies for ESD were 0.6809 for ESD*1 and 0.3191 for ESD*2, (∑χ2 = 0.8586 and P ≥ 0.50); gene frequencies of ACP were 0.2009 for ACP*A, 0.7991 for ACP*B, (∑χ2 = 1.7892 and P > 0.20), with good agreement between the observed and the expected values.
A World Wide Web (WWW) server database engine for an organelle database, MitoDat
Authors: Lemkin Peter F. , Chipperfield Michael , Merril Carl , Zullo Steven 
Published: May 2005

AbstractWe describe a simple database search engine “dbEngine” which may be used to quickly create a searchable database on a World Wide Web (WWW) server. Data may be prepared from spreadsheet programs (such as Excel, etc.) or from tables exported from relational database systems. This Common Gateway Interface (CGI‐BIN) program is used with a WWW server such as available commercially, or from National Center for Supercomputer Algorithms (NCSA) or CERN. Its capabilities include: (i) searching records by combinations of terms connected with ANDs or ORs; (ii) returning search results as hypertext links to other WWW database servers; (iii) mapping lists of literature reference identifiers to the full references; (iv) creating bidirectional hypertext links between pictures and the database. DbEngine has been used to support the MitoDat database (Mendelian and non‐Mendelian inheritance associated with the Mitochondrion) on the WWW.
Identification of proteins by their amino acid composition: An evaluation of the method
Authors: Golaz Olivier , Wilkins Marc R. , Sanchez Jean‐Charles , Appel Ron D. , Hochstrasser Denis F. , Williams Keith L. 
Published: May 2005

AbstractExpression of different genomes can be studied by high‐resolution two‐dimensional electrophoresis (2‐D PAGE). To help these studies, two‐dimensional reference maps of different biological tissues and fluids have been built and can be found in the SWISS‐2DPAGE database, accessible via the World Wide Web network on the ExPASy molecular biology server. Different techniques were used to identify the polypeptides. At the present time, the method considered to be the fastest and the most cost‐effective is amino acid composition analysis (AAC). Proteins, transferred onto polyvinylidene (PVDF) membranes, were submitted to vapor‐phase hydrolysis, derivatized with 9‐fluorenylmethyl chloroformate (FMOC) and separated on an ODS‐Hypersil column. Identification was obtained by using the program ‘AACompIdent’ available from ExPASy. In this work, different experimental parameters, such as contamination, reproducibility and accuracy, have been assessed. First, it has been found that a major source of contamination was human keratin. Next, amino acids have been classified into ‘reliable’ and ‘nonreliable’. Accordingly, ‘bias’ and ‘weights’ were defined for each amino acid, which could be set in the ‘AACompIdent’ program. Finally, examples of identification, including the use of Edman degradation sequence tagging, are described.
Characterisation of proteins from two‐dimensional electrophoresis gels by matrix‐assisted laser desorption mass spectrometry and amino acid compositional analysis
Authors: Wheeler Colin H. , Berry Sophie L. , Wilkins Marc R. , Corbett Joseph M. , Ou Keli , Gooley Andrew A. , Humphery‐Smith Ian , Williams Keith L. , Dunn Michael J. 
Published: May 2005

AbstractAmino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two‐dimensional electrophoresis (2‐DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2‐DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2‐DE gels.
Identification of different galectins by immunoblotting after two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients
Authors: Lutomski Didier , Caron Michel , Cornillot Jean‐Denis , Bourin Philippe , Dupuy Catherine , Pontet Michel , Bladier Dominique , Joubert‐Caron Raymonde 
Published: May 2005

AbstractVertebrate soluble β‐galactoside‐binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross‐reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), even when followed by immunoblottin. However, their different isoelectric points allow their identification using isoelectric focusing and two‐dimensional (2‐D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two‐dimensional electrophoresis with immobilized pH gradient (IPG‐Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2‐D immunoblotting using anti‐galectin 1 (Gal‐1) and anti carbohydrate‐binding protein 15 (CBP15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG‐Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside‐binding proteins even at basic p/s.
Plasma protein adsorption patterns on liposomes: Establishment of analytical procedure
Authors: Diederichs Julia E. 
Published: May 2005

AbstractAfter intervenous (i.v.) injection, colloidal drug carriers such as liposomes, emulsions, polymeric or solid lipid nanoparticles immediately interact with plasma proteins. The adsorbed plasma protein patterns depend on physicochemical characteristics of the carriers' surface and are regarded as a key factro for the in vivo behavior of the carriers. The comprehension of the correlation between protein adsorption and in vivo organ distribution can be utilized to obtain drug targeting to different tissues. Carriers with different protein adsorption patterns will interact with different tissue‐specific receptors or will be recognized by different macrophage subpopulations. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was applied to determine the protein adsorption pattern on polystyrene particles as model carriers. To transfer this analytical method to i.v. injectable colloidal carriers such as liposomes, a new sample preparation method was developed. The separation of liposomes from plasma after incubation was achived by gel filtration using a Sepharose 2B column. This technique allowed a mild separation independent from eluent composition and only according to size differences. Possible protein desorption from the liposomes and adsorption onto the gel were minimized by using an eluent with a sufficiently high ionic stregth. To estimate the efficiency of separation, the content of liposomes and plasma in each fraction being eluted was determined by ultraviolet (UV) spectroscopy. With this new separation method plasma protein adsorption patterns on liposomes could be analyzed for the first time. The sample preparation by gel filtration seemed to have no influence on liposome stability as far as size distribution is concerned.
Pulsed‐field trapping electrophoresis: A computer simulation study
Authors: Desruisseaux Claude , Slater Gary W. 
Published: May 2005

AbstractExperimental investigations have shown that adding a large, globular and neutral protein (such as streptavidin) at one end of the DNA fragments to be separated by gel electrophoresis strongly affects the dynamics of these molecules, leading to what is known as trapping electrophoresis (TE). In TE, the velocity decreases much more rapidly with DNA molecular size than under normal gel electrophoresis conditions, suggesting that TE may be used to increase the power of separation of polyacrylamide gel electrophoresis. Unfortunately, the bands are broader and fewer readable bands can fit on a single gel slab. Our previous theoretical study of TE also predicted the existence of long‐lasting anomalous regimes where one cannot define a velocity or a diffusion constant. These secondary effects of trapping are related to the very broad distribution of detrapping times (the time needed to exit a trap). In order to increase the usefulness of TE, it has been suggested that pulsed fields may help the molecules exit traps more rapidly. In this article, we present a detailed numerical study of pulsed field TE. We conclude that simple pulsed fields alone may not be enough to increase the sequencing power of polyacrylamide TE because the rate of band broadening cannot be controlled. We also report the existence of anomalous regimes in the presence of pulsed fields, a factor that has been previously neglected in analytical models. Other approaches are also proposed.
Influence of optical probing with YOYO on the electrophoretic behavior of the DNA molecule
Authors: Carlsson Christina , Larsson Anette , Jonsson Mats 
Published: May 2005

AbstractThe influence of the fluorescent dye YOYO (1,1′‐(4,4,8,8,‐tetramethyl‐4,8‐diazaundecamethylene)bis[4‐[[3‐methyl‐benzo‐1,1‐oxazol‐2‐yl]methylidene]‐1,4‐dihydroquinolinium] tetraiodide) on the electrophoretic behavior of the DNA molecule was investigated. This is important when using YOYO as a probe in capillary electrophoresis or in fluorescence microscopy studies of DNA with the purpose of studying the migration mechanism of DNA on the molecular level. We have measured the mobility and orientation dynamics (using the linear dichroism technique) for both pure DNA and the YOYO‐DNA complex in agarose gel in order to compare their electrophoretic properties. Mobility decreases, the degree of orientation becomes lower, and the orientational dynamics slower, when YOYO binds to DNA. However, the dependence on field strength of the mobility, orientation and orientational dynamics, are similar for DNA and YOYO‐DNA, indicating that the mode of migration does not change significantly upon binding YOYO to DNA. Furthermore, since our results show that the effect of YOYO on both the degree of orientation and orientational dynamics of the DNA can be measured and therefore be compensated for, it can be concluded that YOYO is an excellent optical probe for the study of the migrational behavior of DNA.
An analysis of the dynamic range and linearity of an infrared DNA sequencer
Authors: McIndoe Richard A. , Hood Leroy , Bumgarner Roger E. 
Published: May 2005

AbstractWe are currently developing genotyping software and protocols for use on the Li‐Cor model 4000(L) infrared fluorescence DNA sequencers. During the development of the genotyping software it became apparent that the potential dynamic range of the instrument was not being realized when the data collection parameters were optimized to produce a high‐contrast image on the computer screen. In particular, we were unable to obtain peaks with signal to noise (S/N) greater than about 30:1 and the stronger peaks often saturated the detector. Because the numerical dynamic range available in 16‐bit data collection mode exceeds 65000, our limited dynamic range of about 30 in actual data was somewhat puzzling. Hence, we undertook a study to explore the dynamic range and linearity of the Li‐Cor DNA sequencer in order to minimize background fluorescence and noise as well as maximize the available S/N. Data is presented on the background and noise using different polyacrylamide gel mixes with various signal gain and offset values, and the relative contribution of the glass, gel and instrumentation to the background fluorescence is discussed. Based on these results, optimum gain and offset values were determined that maintain the background fluorescence at approximately 1–2% of the maximum dynamic range with a minimum amount of noise. Using these optimized values, we determined the detection system is linear over three orders of magnitude by titrating known quantities of an infrared fluorophore‐labeled primer. In addition, we were able to detect approximately 15.2 amoles of labeled primer. The results provided by this study establish a set of guidelines for evaluating how to set the signal gain and offset in order to minimize the background and maximize the S/N and dynamic range of the Li‐Cor sequencer.
Precast commercial polyacrylamide gels for separation of DNA amplificates by temperature gradient gel electrophoresis: Application to clonality analysis of lymphomas
Authors: Suttorp Meinolf , Von Neuhoff Nils , Tiemann Markus , Dreger Peter , Schaub Jürgen , Löffer Helmut , Parwaresch Reza , Schmitz Norbert 
Published: May 2005

AbstractThe third complementary determining region (CDR‐III) of the rearranged immunoglobulin heavy chain (IgH) genes represents a unique marker for a lymphocyte and its clonal descendants and can be amplified by the polymerase chain reaction (PCR) technique. This approach has markedly enhanced the sensitivity for detection of clonal lymphocyte populations in patients with malignant B‐lymphoid neoplasias. To monitor minimal residual disease (MRD) in tissue specimens during or after antineoplastic treatment, the problem of detecting the presence of a few clonal (malignant) lymphocytes in coexistence with a majority of polyclonal lymphocytes has to be addressed. Semi‐nested PCR amplification of CDR‐III rearrangements from specimen infiltrated by tumor cells generates clonal signals in front of a polyclonal background, and therefore high resolution electrophoretic techniques for separation of DNA fragments are required. Temperature gradient gel electrophoresis (TGGE) resolving DNA homo‐ and heteroduplexes according to their thermal stability has been successfully applied for this purpose using special electrophoretic equipment. We describe an adjustment to this technique by using a commercially available precast 0.5 mm thick polycarylamide gel and by changing a standard horizontal electrophoretic device into a TGGE device. By this means we screened patients with B‐cell lymphoma undergoing high‐dosage radiochemotherapy followed by autologous transplantation for continuous presence of clonal (tumor‐specific) CDR‐III rearrangements. Specimens from blood and bone marrow were collected on diagnosis as well as before and after autologous transplantation. In addition, the autograft (bone marrow or peripheral blood hematopoietic stem cells) was analyzed. Tumor cells were easily detected in the transplants and in specimens collected during follow‐up examinations. The clinical value of these findings remains unclear as yet because the number of cases investigated was small and the follow‐up time is still too short. However, we conclude that the technique of combining the sensitivity of PCR with the specificity of high resolution TGGE is easy to use, making it possible to handle, in a clinical routine, a great number of samples within a short time in order to monitor MRD in patients with B‐cell neoplasias.
High resolution slab gel electrophoresis of 8‐amino‐1,3,6‐pyrenetrisulfonic acid (APTS) tagged oligosaccharides using a DNA sequencer
Authors: O'Shea Michael G. , Morell Matthew K. 
Published: May 2005

AbstractA novel electrophoretic method for the analysis of oligosaccharides using DNA sequencer technology is illustrated using malto‐oligosaccharide distributions obtained following isoamylase disgestion of glycogen, wheat starch and potato starch. The debranched starches were derivatized at the reducing end with the charged fluorophore 8‐amino‐1,3,6‐pyrenetrisulfonic acid (APTS). This highly reproducible method provides baseline resolution of oligomers from chain lengths of 3 to more than 80 glucose units, and exhibits high sensitivity with detection thresholds of one femtomole per resolved band. In addition, the reductive amination procedure attaches a single fluorophore per oligosaccharide, allowing calculation of the results on either a mass or a molar basis. The efficacy of the method is illustrated through the determination of the profile of individual oligosaccharides of chain length with a degree of polymerization (DP) < 80, derived from loading less than 15 ng per analysis of glycogen, wheat and potato starches. While the results obtained were superior in resolution and sensitivity to previously reported observations using a range of techniques, they were nonetheless consistent with the overall differences between these polysaccharides. The resolution, sensitivity, reproducibility and high throughput of the method provides substantial advantages over existing methods for the analysis of linear oligosaccharide chain length distributions.
Pyrogallol red‐molybdate: A reversible, metal chelate stain for detection of proteins immobilized on membrane supports
Authors: Shojaee Negin , Patton Wayne F. , Lim Mark J. , Shepro David 
Published: May 2005

AbstractCertain metal complexes selectively interact with proteins immobilized on solid‐phase membrane supports to form brightly colored products. The metal chelates form protein‐dye complexes in the presence of metal ions at acidic pH but are eluted from the proteins by immersing membranes in a solution of basic pH that contains other chelating agents. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, N‐terminal and internal protein sequencing. Among the metal complexes evaluated to date, the triazine dye‐ferrous complexes (ferene S, ferrozine) and the ferrocyanide‐ferric complexes provide the most sensitive detection of proteins immobilized on membranes. While the pyrogallol redmolybdate complex is commonly used in solution‐based total protein assays, its utility as a reversible stain for proteins immobilized on membranes has not been reported. Pyrogallol red‐molybdate complexes readily stain proteins on nitrocellulose and polyvinyl difluoride membranes with similar sensitivity as ferrozine‐ferrous complexes. Analysis of charge‐fractionated carrier ampholytes and synthetic polymers of different L‐amino acids indicate that binding is prominently via protonated α and ε‐amino side chains. Carbamylation of amino groups in bovine serum albumin substantially diminishes pyrogallol red‐molybdate binding to the protein. The stain is reversible, resistant to chemical interference, and compatible with immunoblotting.