bluuebunny/crossref_metadata_2025_split huggingface dataset:
High‐sensitive fluorescent DNA sequencing and its application for detection and mass‐screening of point mutations
Authors: Hattori Masahira , Yoshioka Katsuji , Sakaki Yoshiyuki 
Published: May 2005

AbstractWe describe a rapid and sensitive DNA sequencing method for an automated fluorescent DNA sequencer (AFDS) and its application for detection of point mutations. The method is based on an improved cycle sequencing procedure in which only 10–50 fmol of template DNA is required. Furthermore, it is able to use crude DNA preparation as a template as well as the purified one. Thus, the improved method provided a simplified procedure for sequencing of various types of DNA, including cosmid DNA, in which purification steps were unnecessary. We also developed a novel system for detection of point mutations using AFDS. A set of four lanes is used for the parallel analysis of single‐base profiles of four different samples, instead of for the four‐base profile of a sample. The AFDS exhibits the base profiles of the samples with four different colors in the analyzed data, which enables us to identify a mutation as an additional peak with a color specific for the lane. The feasibility of our system was tested by analyzing polymerase chain reaction (PCR)‐amplified genomic DNAs from four individuals including a carrier of a mutation of C to T. The mutation was clearly identified as an additional “T” peak of a color specific for the carrier. The mutation was also detectable even if 16 individuals including the carrier were simultaneously analyzed on a set of four lanes (four individual samples for each lane). Thus, the novel system is useful for simultaneous detection of mutations in a large number of individual samples.
Manual sequencing using pulsed field
Authors: Turmel Chantal , Brassard Eric , Noolandi Jaan 
Published: May 2005

AbstractThe use of pulsed fields in manual sequencing opens up the compression zone found with a DC field and extends the range of resolution from a few hundred bases to several thousand bases. The band inversion problem is overcome with the proper pulsing conditions, and the bands are sharper than for the DC field case. Accurate visual reading is possible up to about 800–900 bases. The method is compatible with automation techniques, since the band spectrum is stretched continuously during migration, and the smaller fragments are run off the gel.
A PacI/SwaI map of the Pseudomonas aeruginosa PAO chromosome
Authors: Römling Ute , Tümmler Burkhard 
Published: May 2005

AbstractThe PacI/SwaI macrorestriction map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was constructed by fragment size comparisons of complete single and double digests and hybridization with probes of known genomic localization. P. aeruginosa PAO contains five recognition sites for PacI and six sites for SwaI. An integrated DpnI/PacI/SpeI/SwaI map was obtained by fragment pattern and Southern blot hybridization analyses of all combinations of double digestions and by two‐dimensional pulsed‐field gel electrophoresis mapping protocols.
Identification of peaks in capillary zone electrophoresis based on actual mobilities
Authors: Vespalec Radim , Gebauer Petr , Boček Petr 
Published: May 2005

AbstractA procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from −56 to −20 × 10−9 m2V−1s−1 and chloride as the ion modelling the effect of the sample matrix.
Atrazine and simazine determination in river water samples by micellar electrokinetic capillary chromatography
Authors: Desiderio Claudia , Fanali Salvatore 
Published: May 2005

AbstractA rapid and sensitive micellar electrokinetic capillary chromatography analytical method was used for the determination of chlorotriazine herbicides in river water samples. Several electrolyte systems in the pH range 7–10 were tested in order to optimize the separation. The two compounds were separated in less than 10 min and the determination limit was about 0.4 ppb for each herbicide. Recovery values of the method were in the range 80%–117%.
Applications of an automated apparatus for two‐dimensional electrophoresis, Model TEP‐1, for microsequence analyses of proteins
Authors: Nokihara Kiyoshi , Morita Naoki , Kuriki Tomoko 
Published: May 2005

AbstractTwo‐dimensional electrophoresis has become a rapid and powerful separation method for the preparation of samples for sequence analysis depending on the development of automated microsequencers accompanied by a blotting technique. We have constructed an automated apparatus for two‐dimensional electrophoresis, Model TEP‐1, which allows precise analytical runs under computercontrol with a fully automated transfer from the first‐ to the second‐dimensional gel. In the present paper we describe several applications of TEP‐1 followed by blotting and sequencing to reveal the primary structure of proteins in a complex mixture. Several known and unknown proteins were analyzed. Enzymatic digests of proteins could be directly applied onto the TEP‐1, in which a gel of lower molecular weight proteins was used as the second dimension. Subsequent blotting and sequencing provided internal sequence information. The use of TEP‐1, followed by blotting, provides spots containing ca. 10–100 pmol protein. These figures permitted the identification of 10–40 amino acid residues from the N‐terminus. The TEP‐1 provides a simple and rapid separation of complex mixtures obtained from natural sources and chemical, biological or genetic products.
Equipment for rapid homogenization of high numbers of plant tissue for electrophoretic analysis of proteins
Authors: van den Berg Bartel M. , Burg Hendrik C. J. , Tamboer Jan H. A. , Grapendaal Bob 
Published: May 2005

AbstractA homogenizing system was developed to process multiple samples of various plant tissues. The equipment consists of a motor‐driven stainless steel homogenizer that fits perfectly into a hole of a disposable plate. Hard plant material like melon or squash seeds (maximal length of 1.4 cm) could be homogenized within 10 s by forcing the homogenizer towards the bottom of a 24‐ or 48‐well tissue culture plate. Smaller seeds could be homogenized within 10 s in a similar way using a 96‐well tissue culture plate. Leaf tissue, or other types of soft plant tissue, could be homogenized with the same equipment. For homogenization of small samples, e.g. pistils of small flowers, a 60‐well Terasaki plate was used. For homogenization of several hundreds of leaf tissue samples, an apparatus was developed that contains 12 homogenizers driven simultaneously by an electromotor. A tissue culture plate containing 96 leaf samples could be handled in this way in less than 10 min. The performance of the equipment was evaluated by analyzing homogenates of several types of plant tissue by different electrophoretic techniques.
An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase
Authors: Fieldes Mary Ann 
Published: May 2005

AbstractWhen plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD‐dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)‐mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS‐mediated reduction of NBT and for the peroxidase‐catalyzed NADH‐dependent formation of H2O2. This peroxidase‐catalyzed reaction has been proposed for the plant peroxidases involved in lignification.
Effects of bile salt exposure on pancreatic duct barrier function and protein release determined by two‐dimensional electrophoresis and lectin‐affinoblotting
Authors: Hänsler Marion , Arendt Thomas , Lange Kay 
Published: May 2005

AbstractWe analyzed effects of a bile salt on pancreatic duct barrier function and protein release into pancreatic juice in cats, using two‐dimensional‐electrophoresis and lectin‐affinoblotting. Preanalyzed pancreatic juice was perfused along the pancreatic duct following duct exposure to a bile salt. In this case, pancreatic duct mucosa showed a partial loss of barrier function to the leakage of exocrine pancreatic proteins dependent upon molecular weight and charge. The release of a sialic acid possessing protein into pancreatic juice was observed.
The use of radioactive bacteriophage proteins as X–Y markers for silver stained two‐dimensional electrophoresis gels and quantification of the patterns
Authors: Gersten Douglas M. , Ramagli Louis S. , Johnston Dennis A. , Rodriguez Lewis V. 
Published: May 2005

AbstractWe have demonstrated the feasibility of using bacteriophage ghost proteins, tritiated by metabolic labeling, as a set of standard markers for two‐dimensional gels in which the proteins are to be detected by silver staining. The results indicate that a 2.5 μg load of phage proteins yields a reproducible silver pattern of 48 spots. The spots can also be readily identified by radioautography and radiofluorography, establishing their value as a standard constellation of markers. Quantification of these patterns by computerized densitometry indicates a general agreement between detection by silver staining and detection by radiofluorography.
Wheat gliadin: Digital imaging and database construction using a 4‐band reference system of agarose isoelectric focusing patterns
Authors: Black Jeffrey A. , Waggamon Kimberly A. 
Published: May 2005

AbstractAn isoelectric focusing method using thin‐layer agarose gel has been developed for wheat gliadin. Using flat‐bed units with a third electrode, up to 72 samples per gel may be analyzed. Advantages over traditional acid polyacrylamide gel electrophoresis methodology include: faster run times, nontoxic media, and greater sample capacity. The method is suitable for fingerprinting or purity testing of wheat varieties. Using digital images captured by a flat‐bed scanner, a 4‐band reference system using isoelectric points was devised. Software enables separated bands to be assigned pI values based upon reference tracks. Precision of assigned isoelectric points is shown to be on the order of 0.02 pH units. Captured images may be stored in a computer database and compared to unknown patterns to enable an identification. Parameters for a match with a stored pattern may be adjusted for pI interval required for a match, and number of best matches.
On the use of dimension‐less parameters in acid‐base theory. I. The buffer capacity of simple ampholyte solutions
Authors: Rilbe Harry 
Published: May 2005

AbstractA complete mathematical account of the buffer capacity of bivalent ampholytes is given. The consistent use of dimensionless parameters makes it possible to obtain very neat expressions and equations. Direct proof of the well‐known condition K1 ⩾ 4 K2 is presented. Since water is also an ampholyte, its buffer capacity is considered as well.
Polyhydroxy and polyethyleneglycol (meth)acrylate polymers: Physical properties and general studies for their use as electrophoresis matrices
Authors: Zewert Thomas , Harrington Michael 
Published: May 2005

AbstractA new series of materials have been tested for their suitability as electrophoresis matrices. The mechanical and optical properties of gels composed of polyethyleneglycol (meth)acrylate esters or polyhydroxy (meth)acrylate esters in water and in various concentrations of organic solvents are described. Several crosslinkers including polyethyleneglycol and polyhydroxy di(meth)acrylates, piperazine diacrylate, and bisacrylamide were used in these studies. Electrophoretic migration and separation of a series of protein standards through polyethyleneglycol methacrylate (PEGM) 200, PEGM 400, and glyceryl methacrylate is demonstrated. Further, copolymerization of all of the monomers with acrylamide was performed and the distribution of monomer incorporation into the polymer network calculated. All monomers and copolymers that were examined by IR spectroscopy showed greater than 99% polymerization. These results justify their further study for biomolecule separations.
Identification of Gram‐negative bacteria by isoelectric focusing of soluble cellular proteins
Authors: Margull Andreas , Süssmuth Roland , Schulz Peter 
Published: May 2005

AbstractStrains of Escherichia coli. Pseudomonas aeruginosa and Proteus mirabilis, isolated from clinical specimens were disrupted by repeated freezing in liquid nitrogen and thawing at room temperature. The samples were separated by isoelectric focusing using polyacrylamide gels. The resulting protein patterns showed clear differences between the three species and made identification possible.
A rapid periodic acid‐Schiff staining procedure for the detection of glycoproteins using the phastsystem
Authors: Van‐Seuningen Isabelle , Davril Monique 
Published: May 2005

AbstractA procedure for the analysis of glycoproteins and glycopeptides using the PhastSystem with detection by the periodic acid‐Schiff stain is described. Following sodium dodecyl sulfate or nondenaturing polyacrylamide gel electrophoresis and also isoelectric focusing, samples are stained directly for the presence of carbohydrates. By using the PhastSystem, the method is rapid, sensitive, reliable and allows storage of the gels without a change in the stain. As little as 0.1 μg of protein‐associated carbohydrates can be detected. The staining procedure is also used following isoelectric focusing of mucin‐derived glycopeptides to visualize their charge differences and the increase of their isoelectric point after neuraminidase treatment.
Transverse pore gradient gel electrophoresis of lipoprotein [a] using methylenebisacrylamide gradients
Authors: Smejkal Gary B. , Hoff Henry F. 
Published: May 2005

AbstractWe describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linear N,N′‐methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proportions.
Low fluorescence background electroblotting membrane for DNA sequencing
Authors: Chu Tun‐Jen , Caldwell Karin D. , Weiss Robert B. , Gesteland Raymond F. , Pitt William G. 
Published: May 2005

AbstractA low fluorescence background polypropylene (PP) membrane has been developed for ultimate use as an electroblotting membrane in DNA sequencing based on fluorescence detection. The DNA binding capacity of this membrane is improved by a surface modification using radio frequency plasma discharge (RFPD) in ammonia gas. The RFPD operational parameters are evaluated both in terms of membrane nitrogen content and in terms of the product's capacity for binding radioisotope‐labeled DNA fragments. The surface morphologies of the derivatized membranes are examined by scanning electron microscopy; their mechanical and electrical properties, which are important for the subsequent sequencing procedures, are likewise established. Due to the goal of developing a membrane suitable for multiplex processing, in which the electroblotted DNA must withstand dozens of hybridization/stripping cycles, special attention is given the covalent attachment of DNA to the membrane. The modified PP membrane is evaluated in a multiplex sequencing application using radioisotope‐labeled DNA probes, and found to yield somewhat better binding of a given amount of electroblotted DNA than the commonly used GeneScreen membrane. A tenfold repetition of the probing indicates little loss of signal; the membrane‐bound DNA is stable upon storage and shows no detectable loss in probing efficiency after one month.
Oligonucleotide fingerprinting of plant and fungal genomes: A comparison of radioactive, colorigenic and chemiluminescent detection methods
Authors: Bierwerth Susan , Kahl Günter , Weigand Franz , Weising Kurt 
Published: May 2005

AbstractDigoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences were introduced into nonradioactive fingerprint analysis of plant and fungal DNA. The fragment patterns, obtained by blot hybridization of TaqI‐restricted DNA from chickpea (Cicer arietinum) and its fungal pathogen Ascochyta rabiei with digoxigenated probes and either a colorigenic or a chemiluminescent detection method, were compared to those obtained with 32P‐labeled probes. In combination with alkaline phosphatase and its chemiluminescent substrate 3‐(2′‐spiroadamantane)‐4‐methoxy‐4‐(3″‐phosphoryloxy)phenyl‐1,2‐dioxetane (AMPPD) digoxigenated oligonucleotides yielded clear‐cut fingerprints with high signal‐to‐background ratios within several minutes of exposure to X‐ray films. The chemiluminescence reaction remained stable for at least two weeks. A comparison of banding patterns obtained by radioactive versus digoxigenin‐based hybridization and detection techniques revealed substantial differences in the relative signal intensities of bands. Both nonradioactive techniques show a tendency to “equalize” band intensity differences. Whereas 32P‐labeled oligonucleotides are also applicable to in situ hybridization with DNA immobilized in dried agarose gels, gel hybridization did not work efficiently with digoxigenated probes and either substrate.
Impact of separation capacity on the isotachophoretic analysis of organic acids in human seminal plasma and prostatic fluid
Authors: Oefner Peter J. , Häfele Robert , Eberle Johannes 
Published: May 2005

AbstractIn the present study, the impact of pH of both the leading electrolyte and the sample on the separation capacity of an isotachophoretic system, used for the analysis of organic acids in human seminal plasma and prostatic fluid, was assessed. Generally, maximum separable amounts of eleven equimolar pairs of anions decreased within a pH range of 2.90–4.25 of the leading electrolyte due to increasing net mobilities of the analytes with rising pH. The values obtained under equimolar conditions were also valid for nonequimolar mixtures as long as the buffer capacity of the leading electrolyte was sufficient to maintain constant mobilities of the ionic species to be separated throughout the capillary. However, when the sample injected has a buffer capacity of its own and a pH higher than that of the leading electrolyte, separation capacity will vary. For that reason, the maximum separable amount of an aqueous solution of phosphate and citrate was higher than that obtained for seminal plasma. Hence, the injection volume had to be reduced in order to ensure complete resolution. Besides phosphate and citrate, which was the prime component of prostatic fluid, lactate, aspartate and glutamate could be quantitatively determined in seminal plasma with a precision of ± 1.5%.
Use of principal components analysis for mutation detection with two–dimensional electrophoresis protein separations
Authors: Taylor John , Giometti Carol S. 
Published: May 2005

AbstractThe application of two‐dimensional electrophoresis (2‐DE) to mutation detection requires the capability to monitor each protein in a 2‐DE pattern for significant changes in abundance indicative of a mutation event. Previously, mutation searches were done using a univariate outlier detection method in which each protein spot was considered independently in a classical outlier search. An alternative approach to analysis of 2‐DE patterns for quantitative changes is a multivariate procedure which takes advantage of the observation that protein spots in a 2‐DE pattern often represent correlated rather than independent measurements. We have compared the efficiency of univariate and multivariate procedures for mutation detection using data from the Argonne National Laboratory 2‐DE database of mouse liver proteins. Analyses involving a total of over 1500 gels were performed to compare the performance of a multivariate method based on principal components analysis (PCA) with the univariate method. Up to 279 spots from each pattern were used for PCA. First, a simulation was performed to assess the detection efficiency of PCA for single protein spots decreased in abundance by 50%. Then, the ability to detect actual mutations was tested using eight confirmed mutations. Results show that, compared to a univariate approach to analysis of data from the mouse model system, the multivariate method increases the number of protein spots on each 2‐DE pattern that can be monitored for quantitative changes indicative of mutations by compensating for variables that contribute to the background quantitative variability of protein spots.
Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti‐Borrelia burgdorferi IgG antibodies
Authors: Cruz Mabel , Sidén Åke 
Published: May 2005

AbstractAnti‐Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4–10 and pH 4–7 gels. After focusing, the anti‐B.‐burgdorferi antibodies were blotted by affinity‐driven transfer to antigen‐coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG‐IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti‐B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.