bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunoblotting of Yersinia plasmid‐encoded released proteins: A tool for serodiagnosis
Authors: Cremer Josef , Putzker Michael , Faulde Michael , Zöller Lothar 
Published: May 2005

AbstractThe plasmid‐encoded, released proteins (RPs) of Yersinia enterocolitica seroypes 09 and 03 were separated by sodium dodecyl sulfate (SDS)‐pore gradient gel electrophoresis. The RP‐patterns of both serotypes proved to be identical. Five major proteins of Mr 27000, 34700, 35600, 45800, and 46800 were detected. Spontaneously plasmid‐cured derivatives of the two serotypes lost the feature of protein release. By immunoblotting of RP with sera from patients suffering from acute Yersinia infections, specific and reproducible band patterns were obtained. Laser scan densitometry was applied to record the immunoreactions quantitatively. Predominant bands were detected at an Mr of 34700 and 35600. IgA and IgM antibodies appeared as acute‐phase markers rapidly decreasing in the reconvalescent phase. In contrast, immunoblots of patients with supposed chronic yersiniosis were characterized by a persisting IgA and elevated IgG reactivity. The application of RP as diagnostic antigens proved to be advantageous because they are naturally separated from cross‐reacting proteins, common to pathogenic and nonpathogenic strains of Yersinia enterocolitica and Y. pseudotuberculosis.
On the efficiency of methylene blue versus persulfate catalysis of polyacrylamide gels, as investigated by capillary zone electrophoresis
Authors: Caglio Silvia , Righetti Pier Giorgio 
Published: May 2005

AbstractThe efficiency of a novel method of photopolymerization, based on photoinitiating the reaction with methylene blue (MB), in presence of a redox couple (sodium toluenesulfinate and diphenyliodonium chloride), vs. the conventional persulfate ‐ N,N,N′,N′ ‐tetramethylethylenediamine redox couple was investigated as a function of different effectors in solution. Oxygen dissolved in the gelling mixture strongly quenches persulfate catalysis, while leaving essentially unaltered the process initiated by photopolymerization. On the contrary, the presence of 8 M urea substantially accelerates a persulfate‐driven reaction, boosting the conversion of monomers to near completion (> 98%) while leaving the photopolymerization process largely unaffected. Polyacrylamide polymerization has also been performed in a number of hydroorganic solvents (all in a 50 : 50 v/v ratio): dimethyl sulfoxide (DMSO), tetramethyl‐urea, formamide and dimethylformamide. In all cases, the persulfate‐catalyzed reaction was strongly quenched and even completely inhibited (in DMSO), whereas the photopolymerization process was essentially unaffected by any of these organic solvents. The reaction kinetics of the methylene blue‐driven reaction could not be ameliorated when admixing an anionic dye (e.g., eosin Y) to the cationic MB, even when amply changing their molar ratios. Thus, it appears that photocatalysis with MB (and the redox couple sodium toluene‐sulfinate and diphenyliodonium chloride) is a unique process, proceeding at optimum rate under the most adverse conditions, completely insensitive to any kind of positive and negative effectors and able to ensure at least 95% monomer conversion under the standard conditions of 1 h reaction time at room temperature. Due to the fact that the MB system is also unaffected by the pH of the gelling solution (in the pH 4–10 interval), it would appear that such a system is largely superior to any other catalyst mixture so far proposed in the field of polyacrylamide catalysis.
Native polyacrylamide gel electrophoresis of membrane proteins: Glutaminase detection after in situ specific activity staining
Authors: Aledo Juan C. , Gómez‐Biedma Simón , Segura Juan A. , Molina Manuel , de Castro Ignacio Núñez , Márquez Javier 
Published: May 2005

AbstractA new procedure for the analysis and detection of phosphate‐activated glutaminase (EC 3.5.1.2) by native electrophoresis has been developed. The method is based on the in situ detection of glutaminase activity in two different systems of native polyacrylamide gradient gels, containing 3‐(3‐cholamidopropyl)‐dimethyl‐ammonio‐1‐propane sulfonate (CHAPS) or Triton X‐100 as nondenaturant detergents. Crude Triton X‐100 extracts of mitochondria were resolved by electrophoresis. The enzyme was specifically revealed by incubation of the gel with glutamine and coupling the oxidation of the glutamate formed to the reduction of a tetrazolium dye, in the presence of glutamate dehydrogenase trapped in a 1% agar solid overlay. Both Ehrlich ascitic cell and mouse kidney glutaminases were resolved by native electrophoresis and specifically detected with the activity staining. Moreover, the redox‐cycling staining was tested in solution, showing linearity with the amount of glutamate or glutaminase activity present. The method described could be a useful tool for native polyacrylamide gel electrophoresis of membrane proteins.
Apolipoprotein (a): A comparison of isoforms identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis or by sodium dodecyl sulfate‐agarose gel electrophoresis
Authors: Craig Wendy Y. , Poulin Sue E. , Ledue Thomas B. , Kamboh M. Ilyas 
Published: May 2005

AbstractLipoprotein(a) resembles low density lipoprotein in structure, except that a unique apolipoprotein (apo), apo(a), is linked to apo B‐100. Variations in the number of sequence repeats in the apo(a) gene give rise to a range of isoforms. Depending on the method used, 6–30 apo(a) isoforms have been observed; however, the correspondence of these different isoforms has not been reported, making between‐study comparisons difficult. In the present study we address this question by characterizing the apo(a) phenotypes of 48 sera using two previously reported separation methods, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE, 3–12% gels) and SDS‐agarose gel electrophoresis. In addition, the molecular weight of each isoform was estimated using haptoglobin 2–2 polymers as molecular weight standards. Among the 48 sera, 15 distinct apo(a) isoforms were separated by SDS‐PAGE and 28 by SDS‐agarose gel electrophoresis. There was excellent correlation between the two nomenclature systems (r = −0.97, p < 0.001, by rank correlation), and the ranges were totally overlapping, with the same two isoforms being identified as the largest and smallest by either method. The apparent molecular mass range for the isoforms was 294–624 kDa, which is in close agreement with the theoretical molecular mass range of 238–643 kDa, calculated from the sequence and carbohydrate content of recombinant apo(a). The disparity in number of isoforms between methods was expected, due to the poorer separation of apo(a) by SDS‐PAGE; 3.1 ± 1.7 (median, 2.0) SDS‐agarose isoforms were combined for each SDS‐PAGE isoform. The present study demonstrates that the nomenclature systems for apo(a) isoforms separated by SDS‐PAGE or by SDS‐agarose gel electrophoresis are well correlated mathematically and encompass the same size range; however, the better resolution of SDS‐agarose electrophoresis suggests that it is the method of choice for apo(a) phenotyping. As further apo(a) isoforms are identified, it will be important to address the question of a standardized nomenclature, in order to facilitate between‐study comparisons.
Salivary deoxyribounuclease I polymorphism separated by polyacrylamide gel‐isoelectric focusing and detected by the dried agarose film overlay method
Authors: Tenjo Etsuko , Sawazaki Kazumi , Yasuda Toshihiro , Nadano Daita , Takeshita Haruo , Iida Reiko , Kishi Koichiro 
Published: May 2005

AbstractIsoelectric focusing of whole saliva samples on polyacrylamide gels (pH 3.5–5), followed by dried agarose film overlay detection, was employed to determine the type of salivary deoxyribonuclease I (DNase I). Since this detection method had not only a high sensitivity, but also a high band resolution, it was possible to determine DNase I types from saliva samples of 2–5 μL. Pretreatment of saliva samples with neuraminidase simplified the isozyme pattern and enhanced the sensitivity. The DNase I types in all 30 saliva samples showed a good correlation with the types found in the corresponding blood, semen, and urine samples. This preliminary trial indicates that DNase I typing from saliva samples is a new and promising method for individualization of casework samples in the field of forensic biology.
Nonenzymatic extraction of cells from clinical tumor material for analysis of gene expression by two‐dimensional polyacrylamide gel electrophoresis
Authors: Franzén Bo , Linder Stig , Okuzawa Ken , Kato Harabumi , Auer Gert 
Published: May 2005

AbstractWe have compared different methods of preparation of malignant cells for two‐dimensional electrophoresis (2‐DE). We found all methods using fresh tissue to be superior compared to methods using frozen tissue. Our results indicate that nonenzymatic methods of preparation of tumor cells, including fine needle aspiration, scraping and squeezing, have advantages over methods using enzymatic extraction of cells. Nonenzymatic methods are rapid, appear to reduce loss of high molecular protein species, and alleviate the necessity of separating viable and nonviable cells by Percoll gradient centrifugation. Using these techniques, high‐quality 2‐DE maps were derived from tumors of the lung and breast. In the resulting polypeptide patterns, heat shock proteins, non‐muscle tropomyosins and intermediate filament were identified. We conclude that nonenzymatic extraction of malignant cells from fresh tumor tissue improves the possibilities that these techniques may be useful in clinical diagnosis.
Studies on 16 kDa selenium‐containing proteins enriched by means of preparative electrophoresis
Authors: Kyriakopoulos Antonios , Kalcklösch Margrit , Weiß‐Nowak Christian , Behne Dietrich 
Published: May 2005

AbstractBy in vivo labeling with [75Se]selenite and separation of the proteins in the tissue homogenates by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), several selenium‐containing proteins or protein subunits were detected in rat tissues (liver, lung, spleen and prostate). Their distribution among the cell components was investigated after fractionation by means of differential centrifugation. The selenium‐containing proteins in the 16 kDa range were found to be mainly membrane‐bound. By two‐dimensional electrophoresis they were resolved into three labeled spots, two with the same relative molecular mass and pI values of about 4.8 and 5.0 and the third with a slightly lower molecular mass and a pI of 4.8. For further investigation they were concentrated and separeted from the other labeled compounds by SDS‐PAGE using preparative flow‐through electrophoresis.
Our obsession with high resolution in gel electrophoresis: Does it necessarily give the right answer?
Authors: Wrigley Colin W. , Gupta Ram B. , Bekes Ference 
Published: May 2005

AbstractPoor resolution of protein zones in an electrophoretic pattern may not necessarily be the result of poor technique. The example is given of the ‘streak material’, extracted from wheat flour, now recognised to be aggregated subunits of glutenin. The size distribution of the aggregated glutenin ‘streak’ is the key to elucidating the functional properties of wheaten dough. A stepped‐layer gel technique has been devised to quantitate the proportions of aggregated glutenin in specific size groupings.
Control of electrohydrodynamic distortion of sample streams in continuous flow electrophoresis using oscillating fields
Authors: Heller Christoph , Limat Laurent , Sergot Philippe , Viovy Jean Louis 
Published: May 2005

AbstractContinuous flow electrophoresis is a method to separate ions contained in a sample continuously injected into a laminar flow of electrolyte as a cylindrical stream. Usually, the sample is more conductive than the electrolyte, and the charges created at the sample‐electrolyte interface lead to electrohydrodynamic distorsions which reduce the separation power of this technique. We demonstrate theoretically that the rate of electrohydrodynamic distorsion of a cylindrical sample stream can be reduced to zero, by superimposing to the AC field responsible for the separation of a DC field transverse to it and to the flow direction, with an appropriate frequency, and an effective strength equal to that of the DC field. Using a continuous flow electrophoresis chamber, in which such a field is produced using capacitive electrodes, the major predictions of the theory are confirmed. In particular, it is shown that a sample stream more conductive than the carrying electrolyte, which was seriously deformed in the absence of a transverse AC field, recovers its cylindrical shape in presence of the field. The implications of this discovery for the separating power of continuous‐flow electrophoresis are discussed.
Centrifuge‐blotting of proteins after separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Hermansen Leonila F. , Pedersen Øyvind , Sletten Knut 
Published: May 2005

AbstractA protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifugeblotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)‐polyacrylamide gel with 1 M KC1, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut‐off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N‐terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%–30% has been obtained.
Salivary protein interactions with oral bacteria: An electrophoretic study
Authors: Newman Frances , Beeley Josie A. , MacFarlane T. Wallace , Galbraith Julie , Buchanan Luise 
Published: May 2005

AbstractThe relatively low levels of human salivary proteins in whole saliva as compared to the ductal secretions may be related to their interaction with oral bacteria. These interactions are thought to play an important role in the microbial colonisation of the mouth, and salivary proteins adsorbed to oral surfaces have been implicated in adherence. In this study we attempted to investigate the prevalence of interactive strains by screening a range of oral bacteria. Parotid saliva was incubated with bacterial suspensions and consequent alterations to the salivary protein profile determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A number of salivary components were found to be preferentially removed, particularly by those species known to be primary colonisers of clean tooth surfaces.
A nonlinear wide‐range immobilized pH gradient for two‐dimensional electrophoresis and its definition in a relevant pH scale
Authors: Bjellqvist Bengt , Pasquali Christian , Ravier Florence , Sanchez Jean‐Charles , Hochstrasser Denis 
Published: May 2005

AbstractA new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two‐dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on pK values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10–25°C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.
Evolution of coxsackie B virus during in vitro persistent infection: Detection of protein mutations using two‐dimensional polyacrylamide gel electrophoresis
Authors: McLaren John , Argo Evelyn , Cash Phillip 
Published: May 2005

AbstractSerotype 5 coxsackie B virus (CBV5) can establish in vitro persistent infections in human rhabdomyosarcoma (RD) cells. This paper describes the characterisation of the virus released from the persistently infected RD cell line designated piRD‐3673. Although infectious virus was released for 42 sequential passages of piRD‐3673 cells, no gross virus‐specific cytopathic effect was detected when the cells were examined by light microscopy. Two‐dimensional polyacrylamide gel electro‐phoresis was used to compare the virus released from piRD‐3673 cells with the CBV5 isolate (CBV‐3673) used to initiate the persistent virus infection. Two of the virus intracellular proteins (apparent molecular weights 33 000 and 39 000, designated p33 and p39, respectively) increased in their net basic charge for the virus released from piRD‐3673 cells compared to CBV‐3673; a reduction in the apparent molecular weights of p33 and p39 was also observed. The charge alteration for both p33 and p39 was a two‐stage process, the accumulative effect of which resulted in p33 increasing in pI from 6.14 to 6.53 and p39 increasing in pI from 6.29 to 6.63. The first mutation of p33 and p39 occurred between passages 7 and 10 of piRD‐3673 cells and affected both the charge and apparent molecular weight of these two proteins. The second mutation at passage 15 of piRD‐3673 cells caused only a change in the charge of p33 and p39. Two other virus proteins (p54 and p75) showed no evidence of mutation over the same passage history of piRD‐3673 cells. The virus released from piRD‐3673 cells also differed from CBV‐3673 by two further criteria, a reduction in plaque‐forming efficiency in HEp‐2 cells and increased virus replication in RD cells. These data on virus evolution are discussed in relation to the maintenance of persistent CBV infections and the presence of naturally occurring CBV variants.
On the kinetics of photopolymerization: A theoretical study
Authors: Lyubimova Tatyana , Righetti Pier Giorgio 
Published: May 2005

AbstractIn dye‐sensitized polymerization, some paradoxical phenomena have been found, such as an anomalous decrease of reaction rate with decrementing thicknesses of the gelling layers. By mathematical modelling, and experimental verification, it has been found that high incorporation efficiencies (> 95% conversion of monomers into the growing polymer) can only be obtained at the correct levels of dye in the gelling solution and by using the proper light intensity. Paradoxically, if levels of sensitizer or incident light intensities are too high, the rate of photosen‐sitizer consumption is too high, as compared with the rate of monomer incorporation, so that dye depletion occurs prior to chain elongation, and the reaction suddenly comes to a stop. In methylene‐blue catalysis, the curve correlating the monomer incorporation with the catalyst concentration exhibits a maximum, indicating poor conversions both below and above a critical catalyst level. When correctly used, photopolymerization still offers unique advantages over chemical (persulfate) polymerization, such as absence of oxidizing conditions and control at the onset of reaction, while guaranteeing high conversion efficiencies (> 95%).
Use of fluorogenic substrates to visualize trypsin and chymotrypsin inhibitors after electrophoresis
Authors: Weder Jürgen K. P. , Haußner Kristine , Bokor Maria V. 
Published: May 2005

AbstractFluorogenic substrates were tested as a means of increasing both the sensitivity and the selectivity of trypsin and chymotrypsin inhibitor detection after electrophoretic separation. Out of six substrates applied to cellulose acetate membranes, N a‐benzyloxycarbonyl‐L‐arginine‐4‐methylcoumarinyl‐7‐amide (Z‐Arg‐MCA) and benzylxycarbonyl‐glycyl‐glycyl‐L‐arginine‐4‐trifluoromethylcoumarinyl‐7‐amide (Z‐Gly‐Gly‐Arg‐TFMCA) were found to be suitable for trypsin, and L‐alanyl‐L‐alanyl‐L‐phenylalanine‐4‐methylcoumarinyl‐7‐amide (Ala‐Ala‐Phe‐MCA) was suitable for chymotrypsin. A procedure to detect trypsin and chymotrypsin inhibitors, and to discriminate between them, was developed. After electrophoresis, slab gels were first incubated with the enzyme (bovine trypsin, bovine chymotrypsin, or human duodenal juice) at 37°C, and then covered with the respective substrate membrane and incubated at room temperature while being observed under UV light. Dark blue inhibitor bands on a light‐blue‐fluorescent background were obtained with Z‐Arg‐MCA/trypsin and Ala‐Afa‐Phe‐MCA/chymotrypsin, whereas Z‐Gly‐Gly‐Arg‐TFMCA/trypsin resulted in dark inhibitor bands on a fluorescent green background. The “inhibitor overlay membrane technique” (IOM technique) was used after polyacrylamide gel isoelectric focusing with carrier ampholytes and immobilized pH gradients, pore‐gradient polyacrylamide gel electrophoresis, and sodium dodecyl sulfate pore‐gradient polyacrylamide gel electrophoresis.
Restriction landmark genomic scanning method and its various applications
Authors: Hayashizaki Yoshihide , Hirotsune Shinji , Okazaki Yasushi , Hatada Izuho , Shibata Hideo , Kawai Jun , Hirose Kenji , Watanabe Sachihiko , Fushiki Shinji , Wada Seiji , Sugimoto Toshikado , Kobayakawa Kiyoshi , Kawara Tsutomu , Katsuki Motoya , Shibuya Tohru , Mukai Tsunehiro 
Published: May 2005

AbstractWe have developed a new genome scanning method (restriction landmark genomic scanning (RLGS)), based on the new concept of using restriction enzyme sites as landmarks. RLGS employs direct end labeling of the genomic DNA digested with a restriction enzyme and two‐dimensional electrophoresis with high‐resolution. Its advantages are: (i) high‐speed scanning ability, allowing simultaneous scanning of thousands of restriction landmarks; (ii) extension of the scanning field using different kinds of landmarks in an additional series of electrophoresis; (iii) application to any type of organism because of direct‐labeling of restriction enzyme sites and no hybridization procedure; and (iv) reflection of the copy number of the restriction landmark by the spot intensity which enables distinction of haploid and diploid genomic DNAs. The RLGS method has various applications because it can be used to scan for physical genomic DNA states, such as amplification, deletion and methylation. The copy number of the locus of a restriction landmark can be estimated by the spot intensity to find either an amplified or deleted region. The methylation state of genomic DNA can also be discovered by use of a methylation‐sensitive restriction enzyme sites as a restriction landmark (restriction landmark genomic scanning for screening methylated sites, RLGS‐M). This article introduces the basic principle of RLGS and its applications to the analysis of cancer, mouse mutant DNAs and tissue‐specific methylation, showing the usefulness of RLGS for a variety of biological fields.
Two‐dimensional gel selection of protein binding sites on DNA
Authors: Boffini Andrea , Prentki Pierre 
Published: May 2005

AbstractWe have developed a gel electrophoretic approach for visualizing and cloning protein binding sites from complete genomes. This system consists of a simple two‐dimensional band shift, in which protein‐DNA complexes are retarded in the first dimension, performed at low temperature, and disrupted in the second dimesion, performed at high temperature. We present here results obtained with the integration host factor (IHF) and cAMP receptor protein (CRP) proteins of Escherichia coli, and discuss some of the important methodological aspects of the technique.