bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of micellar electrokinetic chromatography to the quality control of pharmaceutical formulations: The analysis of xanthine derivatives
Authors: Korman Milos , Vindevogel Johan , Sandra Pat 
Published: May 2005

AbstractMicellar electrokinetic chromatography (MEKC) has been applied to the analysis of three different drugs. Although belonging to different therapeutic classes, all these drugs contain xanthine derivatives as active substances. Pen‐toxifylline was separated from eight related xanthines. Quantitative MEKC was applied to determine impurities (caffeine and xanthine) in the purified drug at the 0.05–0.1% level and also for the determination of the active substance in Agapurin® tablets. Ethofylline and theophylline were separated from ephedrine and mebrophenhydramine and determined in Xantedrylettae® tablets while caffeine was separated from mephenhydramine and determined in Kinedryl® tablets. In all cases, simple borate buffers with sodium dodecyl sulfate as the surfactant were satisfactory and little separation optimization was required. The relative standard deviation (RSD) of the migration times was better than 1% while the RSD of the observed areas was better than 2%. This demonstrates that MEKC is a valuable alternative for the traditional high‐performance liquid chromatography analysis of drugs and drug formulations.
Influence of structure on the behavior of flavonoids in capillary electrophoresis
Authors: Pietta Piergiorgio , Mauri Pierluigi , Bruno Anna , Gardana Claudio 
Published: May 2005

AbstractIn an attempt to elucidate the relation between structure and electrophoretic behavior, the effect of pH, surfactant and buffer on electrophoretic mobilities and resolution of several flavonoids, differing in structure, has been investigated. Ring‐B substitution and the presence of a free hydroxy group at C7 of ring A play an important role, whereas O‐methoxylation is less significant. Glycosylation also contributes in determining different degrees of complexation with borate, thus affecting the mobilities.
Gel dependence of electrophoretic mobilities of double‐stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis
Authors: Gast Frank‐Ulrich , Sänger Heinz Ludwig 
Published: May 2005

AbstractDouble‐stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B‐form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either ≥ 2.5% agarose or ≥ 8% acrylamide with high crosslinking (19:1 acryl‐amide: N,N′‐methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 ± 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small ≈︁ 70% base‐paired rod‐like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140–230 bp, whereas 130 ± 20 bp can be determined from electron micrographs and 123–126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low‐crosslinked polyacryl‐amide gels (2.6%C) is comparable to the determination by alternative methods.
Quantification of silver‐stained proteins resolved by two‐dimensional electrophoresis: Genetic variability as related to abundance and solubility in two maize lines
Authors: Damerval Catherine 
Published: May 2005

AbstractRelative abundar ce and solubility of proteins from etiolated coleoptiles of maize were investigated using two‐dimensional electrophoresis (2‐D PAGE). Automatic quantification of silver‐stained polypeptides on replicate 2‐D gels made it possible to test the linearity of the relationship between spot integrated optical density and protein amount in the range from 15 μg to 135 μg per gel, in two inbred lines. A linear response was found for more than 60% of the spots in each genotype. When a linear response was found in both lines for a given spot, the slope values were similar in 94% of the cases, indicating the reliability of silver staining for polypeptide quantification. The parameters of the curves allowed the definition of protein classes of different abundances that could be compared for genetic variability between the two lines. From a comparison between standard 2‐D patterns (trichloroacetic acid‐acetone extracted proteins) and patterns obtained from Tris buffer extracted proteins, it appeared that 92% of the proteins visualized in the standard gels were soluble. No difference in genetic variability, ether qualitative or quantitative, was evidenced between the various classes of abundance, or between soluble and insoluble proteins.
Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments
Authors: Haas Hildegard , Budowle Bruce , Weiler Günter 
Published: May 2005

AbstractA discontinuous borate/formate buffer system is presented for horizontal polyacrylamide gel electrophoresis of DNA fragments. The resolution potential of the system could be altered by changing the total monomer concentration (5–9%T), the concentration of the crosslinker piperazine diacrylamide (2–5%CPDA), as well as the concentration of formate in the gel (40–120 mM), the leading ion of the buffer system. The separation of DNA fragments would be improved by increasing the migration distance from 22 to 28 cm. This discontinuous polyacrylamide gel electrophoresis system proved highly reproducible.
An investigation of the factors controlling the staining and destaining of electrophoresis gels
Authors: Hitchman Michael L. , Ekstrom Bjorn 
Published: May 2005

AbstractA general analysis is developed for the effect of mass transport and kinetic parameters on the rate of staining and destaining of electrophoresis gels. The various contributions to these overall processes are discussed and, in particular, the ways in which the rate of solution flow, temperature and gel properties can influence staining procedures are highlighted. It is shown that for reproducible, rapid and uniform staining a rotating gel system, analogous to a rotating disc, provides the necessary controlled laminar flow. The theoretical equations derived are compared with experiments and it is shown that at high gel rotation speeds mass transport in solution does not have a major controlling influence on rates of staining and destaining. The temperature dependence of these rates also suggests that there is no significant control by the rate of interaction between stain and protein molecules. The major controlling factor under these conditions is then concluded to be the transport of stain in the gel itself, and the theoretical analysis and time‐dependent experimental results allow a determination of the corresponding diffusion parameters.
Iron‐Induced conformational change in human lactoferrin: Demonstration by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and analysis of effects of iron binding to the N and C lobes of the molecule
Authors: Ying Li , He Jianglin , Furmanski Philip 
Published: May 2005

AbstractAnalysis of Fe‐saturated‐ and apo‐lactoferrin by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) without heating the samples prior to application revealed a substantial difference in mobility. The mobility shift was fully reversible on repetitive removal and readdition of Fe. Binding of a single Fe to the N‐lobe binding site was sufficient to cause the gel shift; binding of a second Fe to the C‐lobe site did not further alter mobility. Removal of Fe from the N lobe of Fe2 lactoferrin did not restore mobility to the position of apolactoferrin. No change in mobility with Fe binding was detected in N and C lobes isolated from intact lactoferrin by controlled trypsin digestion. The data indicate that a conformational change induced by Fe binding to a single site on lactoferrin is detectable by SDS‐PAGE and that this change requires an intact molecule, possibly due to the need for interactions between the two homologous lobes of the molecule.
Use of antibodies against synthetic peptides for analysis of molecular multiplicity: Human deoxyribonuclease I polymorphism
Authors: Yasuda Toshihiro , Nadano Daita , Takeshita Haruo , Kishi Koichiro 
Published: May 2005

AbstractEight different antibodies were raised in rabbits and chickens by injecting them with two synthetic N‐ and C‐peptides, which corresponded to the N‐ and C‐terminal 15 residues of human deoxyribonuclease I (DNase I). These two peptides were used as immunogens, both free and conjugated with keyhole limpet hemocyanin (KLH). A rabbit antiserum against the C‐peptide conjugated with KLH (rabbit anti‐C/KLH) and two chicken antisera against the C‐peptide itself (chicken anti‐C) and conjugated with KLH (chicken anti‐C/KLH) reacted well with purified DNase I, blotted onto a transfer membrane after isoelectric focusing in a polyacrylamide gel. A clear isoenzyme pattern identical to that detected with a rabbit antiserum against the parent enzyme (rabbit anti‐DNase I) was observed. Surprisingly, the chicken anti‐C antiserum was found to be a powerful and highly specific tool for the determination of urinary DNase I phenotypes (Kishi et al., Hum. Genet. 1989, 81, 295–297) and was not inferior in any respect to the rabbit anti‐DNase I. These findings show that the selection of immune animal is one of the most important factors for producing an effective anti‐peptide antibody. Unfortunately, the anti‐peptide antisera obtained in this study neither blocked DNase I enzyme activity nor did it bind to the enzyme.
Characterization of gene expression in clinical lung cancer materials by two‐dimensional polyacrylamide gel electrophoresis
Authors: Okuzawa Ken , Franzén Bo , Lindholm Johan , Linder Stig , Hirano Takashi , Bergman Thomas , Ebihara Yoshiro , Kato Harubumi , Auer Gert 
Published: May 2005

AbstractFourteen human lung tumors of various histopathological types were subjected to two‐dimensional gel electrophoresis (2‐DE). Samples were prepared for 2‐DE using a nonenzymatic sample preparation (NESP) technique recently established in our laboratory. Variations in the expression of some polypeptides were observed between tumors of different histopathological types. To this end, high expression of β‐tubulin, heat shock proteins 73 and 90, lamin B, and proliferating cell nuclear antigen (PCNA) were observed in small cell lung carcinomas (SCLC). One polypeptide of unknown identity (35 kDa, pI 5.5) was significantly overexpressed in primary lung adenocarcinomas compared with SCLC, squamous cell lung carcinomas, metastatic lung adenocarcinomas from colon and rectum, and normal tissue. The amino acid composition of this polypeptide is presented. In summary, combining the NESP technique and 2‐DE is an effective approach to define tumor‐specific markers.
An examination of the β‐glucosidase (linamarase) banding pattern in flax seedlings using ferguson plots and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis
Authors: Fieldes Mary Ann , Gerhardt Karen E. 
Published: May 2005

AbstractThe reported polyacrylamide gel electrophoresis banding pattern for the main β‐glucosidase (linamarase) component from flax seed consists of five bands, made up of 62.5 and 65 kDa subunits; this component has an estimated molecular weight of 570–670 kDa. The present study used Ferguson plots to estimate the molecular weight of each electrophoretic band, plus two additional bands which were detected. From low to high relative mobility, the seven bands formed a linear series with estimated molecular weights from 1200 to 245 kDa. Each was 160 kDa smaller, and less charged, than the preceding band. This 160 kDa difference between bands did not appear to be consistent with the reported subunit size. Each band produced a corresponding band on sodium dodecyl sulfate (SDS)‐gels. The decreases in molecular weight between the bands on nondenaturing gels and their corresponding bands on SDS‐gels were multiples of the 62–65 kDa value. However, the estimated molecular weights of the SDS bands themselves and of the differences between the SDS bands were, again, not consistent with the proposed subunit size. The results suggest that active forms of this enzyme may contain a second minor component (possibly a 30–35 kDa component) in addition to the 62.5 and 65 kDa subunits.
Protein composition of the human heart: The construction of a myocardial two‐dimensional electrophoresis database
Authors: Jungblut Peter , Otto Albrecht , Zeindl‐Eberhart Evelyn , Pleißner Klaus‐P. , Knecht Mathias , Regitz‐Zagrosek Vera , Fleck Eckart , Wittmann‐Liebold Brigitte 
Published: May 2005

AbstractMolecular changes occurring in myocardial diseases are not well understood. Proteins, as regulatory molecules, should play an important role in the etiology of these diseases. The method of two‐dimensional electrophoresis (2‐DE) allows the analysis of some thousand proteins with one experiment. An important prerequisite for this kind of investigation is the possibility of identifying the proteins separated by 2‐DE. We resolved 3239 proteins of the human myocardium and tried to identify 33 proteins by amino acid analysis and microsequencing. Twenty proteins were identified by search for the proteinchemical data obtained in the Martinsried Institute Protein Sequence Database. Comparisons of 2‐DE patterns of different size, which were obtained in different laboratories, were performed with the result that proteins identified on a 2‐DE map of one laboratory can be assigned to spots of 2‐DE maps produced by another laboratory. Our results show the usefulness of a myocardial 2‐DE database; they can be used in different laboratories and make it possible to generate a collection of important human myocardial proteins in a 2‐DE database for comparative studies worldwide.
Separation and characterization of rice proteins
Authors: Tsugita Akira , Kawakami Takao , Uchiyama Yotaro , Kamo Masaharu , Miyatake Norifumi , Nozu Yuzo 
Published: May 2005

AbstractRice proteins from nine tissues and one organelle (leaf, chloroplast, stem, root, germ, dark germinated seedling, seed, bran, chaff and callus) were isolated and then separated by two‐dimensional gel electrophoresis (2‐DE). The protein spots were characterized according to molecular weight, isoelectric point and partial amino‐terminal sequence. Electrophoresis was carried out by isoelectric focusing (IEF), nonequilibrium pH gradient electrophoresis (NEPHGE) and immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the second dimension. With the aid of nine marker proteins, the patterns of IEF, NEPHGE and IPG 2‐DE gels were graphically combined by computer into a single synthetic image for each tissue, respectively, and these images for the nine tissues and one organelle were again combined into a single 2‐DE image for the integrated rice protein spots. The rice 2‐DE gel image resolved 4892 proteins. About 3% of the spots are characterized by amino‐terminal sequencing.
Use of polyacrylamide gel electrophoresis to detect structural variations in kilobase‐sized DNAs
Authors: Stellwagen Nancy C. 
Published: May 2005

AbstractThe electrophoresis of linear, kilobase‐sized DNA molecules with permuted sequences has been studied in polyacrylamide and agarose gels. Plasmid pBR322, bacteriophage ϕX174, and the SV40 minichromosome were each digested with a series of single‐cut restriction enzymes. The linearized, permuted isomers of all three DNAs exhibit different mobilities in large‐pore polyacrylamide gels, suggesting that all three DNAs contain sites of anisotropic, sequence‐dependent curvature. Various experimental parameters such as acrylamide concentration, crosslinker ratio and buffer composition affect the magnitude of the observed differential mobilities. Band sharpness appears to be optimal in polyacrylamide gels containing 6.9–8.1%T and 0.5–1%C. Only small mobility differences are observed for the linearized, permuted sequence isomers in agarose gels.
The use of coated and uncoated capillaries for the electrophoretic separation of DNA in dilute polymer solutions
Authors: Barron Annelise E. , Sunada Wade M. , Blanch Harvey W. 
Published: May 2005

AbstractWe show that both uncoated and polyacrylamide‐coated capillaries provide separation of large DNA restriction fragments (2.0–23.1 kbp) by capillary electrophoresis in dilute cellulosic polymer solutions. Uncoated capillaries, however, provide significantly better resolution of DNA fragments, particularly when ultra‐dilute polymer solutions are used. This is because electroosmotic flow in uncoated capillaries increases the residence time of DNA in the capillary, without significantly contributing to band‐broadening. At a given field strength and polymer concentration in the buffer, the electrophoretic mobilities of DNA restriction fragments in coated capillaries are virtually identical to those previously measured in uncoated capillaries. It is concluded that the fused silica surface of the capillary does not play a significant role in the mechanism of DNA separation by capillary electrophoresis in uncrosslinked polymer solutions. Thus, the separation of large DNA which has been observed to occur in ultra‐dilute polymer solutions arises primarily from entanglement interactions between the cellulosic polymers and DNA restriction fragments which occur within the bulk of the polymer solution.
Biological activity of prostate‐specific antigen isolated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electroelution
Authors: Tessmer Uwe , Quack Thomas , Donn Folkert , Leuner Astrid , Dernick Rudolf 
Published: May 2005

AbstractHuman prostate‐specific antigen (PSA), a 33 kDa kallikrein‐like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate‐buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS‐PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS‐stable complexes with the two major extracellular protease inhibitors in blood, α1‐antichymotrypsin (α1‐ACH) and α2‐macroglobulin (α2‐M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors α1‐ACH and α2‐M.
Chamber‐specific expression of human myocardial proteins detected by two‐dimensional gel electrophoresis
Authors: Pleißner Klaus‐Peter , Regitz‐Zagrosek Vera , Weise Christoph , Neuß Michael , Krüdewagen Bernhard , Söding Peter , Buchner Klaus , Hucho Ferdinand , Hildebrandt Alfred , Fleck Eckart 
Published: May 2005

AbstractHigh resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), followed by computer‐assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end‐stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2‐D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: Mr/pI:20.5 kDa/4.6; SSP 1119: Mr/pI:20.6 kDa/4.5; SSP 0117:Mr/pI:20.7/< 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: Mr/pI:17.2 kDa,/< 4.4; SSP 0114:Mr/pI:17.6 kDa/< 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2‐D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.
Studies on the carbohydrate moieties of the timothy grass pollen allergen Phl p I
Authors: Petersen Arnd , Becker Wolf‐Meinhard , Moll Hermann , Blümke Maren , Schlaak Max 
Published: May 2005

AbstractTimothy grass pollen was investigated in order to determine the carbohydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N‐glycosylation site was deduced from the cDNA data of Phl p I. After two‐dimensional polyacrylamide gel electrophoresis, followed by blotting of pollen extract and by use of the monoclonal antibody IG 12 we identified at least six isoallergens of Phl p I with the main spots at a molecular mass of 35–37 kDa and a pI range of 6.5–7.3. Deglycosylation by trifluoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatment with N‐glycosidase A resulted in a partial deglycosylation, while N‐glycosidase F and O‐glycosidase had no effect. Ten lectins were investigated for their binding to Phl p I components: Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues), while Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A (indicating mannose, glucose or N‐acetylglucosamine residues) showed weak binding. By neutral sugar analysis we determined similar contents of the monosaccharides in the isoallergens. In order to study the influence of the carbohydrate structures of Phl p I on IgE reactivity we tested some patient sera for their reactivity with intact and deglycosylated Phl p I. Even though most of the IgE antibodies bind at the protein core, we detected one serum that recognized carbohydrate moieties on the Phl p I.