bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrophoretic resolution versus fluctuations of the lateral dimensions of a capillary
Authors: Slater Gary W. , Mayer Pascal 
Published: May 2005

AbstractBecause the local electrical resistance is inversely proportional to the local cross‐section of a capillary, the intensity of the electric field varies along the migration path if the inner diameter of the capillary is not constant. Therefore, fluctuations of the lateral dimensions of a capillary can directly affect the net elution time as well as the peak width, and, hence the final resolution. In this article, we develop the theoretical framework for the study of such effects. We then examine the simple case where both the mobility and the diffusion coefficient are field‐independent; in particular, we demonstrate that resolution can be severely reduced if the inner walls are not flat, and that optimal resolution is always obtained for perfectly flat walls. Generalized to ultrathin gels, our results clearly indicate that both random and systematic variations of the gel thickness can greatly affect the performance of the separation process. Acceptable degrees of flatness are estimated for both geometries. This study thus provides a quantitative understanding of the type of quality control one requires to obtain optimal results with capillaries and ultrathin gels.
Dispersion coefficients of a protein and DNA fragment in polyacrylamide gel electrophoresis as a function of parameters defining the effective gel pore size and particle size
Authors: Chang Huan‐Tsung , Chrambach Andreas 
Published: May 2005

AbstractThe dispersion coefficient, D′, of the representative homogeneous protein, conalbumin, decreases linearly as the polyacrylamide concentration increases from 4 to 14%T (2%C), and varies in a biphasic fashion as %C (Bis) is increased from 2 to 20%, with a broad peak between 5 and 15%C. D′ increases linearly with the concentration of the initiator, potassium persulfate, in the range of 0.01–0.15%. D′ remains constant when the field strength is varied from 5 to 15 V/cm. A DNA fragment (1857 bp) exhibits a constant D′ in 4–6% polyacrylamide (2%C) at a field strength of 1 V/cm, and a linearly increasing D′ at 5 V/cm, in analogy to its previously observed behavior in agarose gels. In solutions of uncrosslinked polyacrylamide, the decrease of the D′ of conalbumin with polymer concentration is not significantly different from that in 2% N,N′‐methylenebisacrylamide‐crosslinked gels in the range of 4–14%T, while the decrease of mobility with polyacrylamide concentration is much steeper in 2% crosslinked compared to uncrosslinked polymer. Finally, –∂ (log D′)/∂T was found to be proportional to the retardation coefficient, KR (= –∂ (log μ)/∂T), in polyacrylamide gels. The ratio of –∂ (log D′)/∂T over KR increases with field strength in the range of 5–15 V/cm.
The determination of nitrite and nitrate in human blood plasma by capillary zone electrophoresis
Authors: Ueda Toshiko , Maekawa Tsuyoshi , Sadamitsu Daikai , Oshita Shuzo , Ogino Keiki , Nakamura Kazuyuki 
Published: May 2005

AbstractThe determination of nitrite and nitrate in human blood plasma by capillary zone electrophoresis (CZE) is described. Electrophoresis was carried out in a 65 cm long, 75 μm wide silica capillary, using 750 mM sodium chloride containing 5% NICE‐Pak OFM Anion‐BT (an additive reversing the electroosmotic flow) as running buffer, at a potential of 20 kV, with on‐column UV detection at 214 nm. Under these conditions, the two anions migrated in the order of nitrite and nitrate, completely resolved from each other. Forty‐one samples of ultrafiltered blood plasma of healthy volunteers were analyzed. The concentrations of nitrite and nitrate were 0.15 ± 0.07 mg/L and 3.2 ± 1.6 mg/L (mean± SD), respectively, with a linear correlation in the range of 0.1–50 mg/L. The within‐day and between‐day coefficients of variation (CVs) for nitrite and nitrate were lower than 5% and 8%, respectively. The recovery was 97‐114%. The estimated plasma volume needed for each determination was 108 nL.
Purification of wheat glutenin subunits by preparative acid and two‐dimensional electrophoresis
Authors: Curioni Andrea , Morel Marie‐Hélène , Furegon Luca , Redaelli Rita , Peruffo Angelo Dal Belin 
Published: May 2005

AbstractReduced alkylated glutenin subunits from wheat flour were fractionated by preparative electrophoresis at acid pH. The high molecular weight glutenin subunits (HMW‐GS) and some of the low molecular wight glutenin subunits (LMW‐GS) were purified by this one‐step procedure, whereas the remainder of the LMW‐GS, comigrating in the acid system, were purified in a second step by electroendosmotic preparative electrophoresis in the presence of sodium dodecyl sulfate. The quantities of recovered protein were sufficient for biochemical characterization and/or antibody production.
Serine protease inhibitor activity of recombinant squamous cell carcinoma antigen towards chymotrypsin, as demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis
Authors: Nawata Shugo , Tsunaga Nagato , Numa Fumitaka , Tanaka Tatehiko , Nakamura Kazuyuki , Kato Hiroshi 
Published: May 2005

AbstractSquamous cell carcinoma (SCC) antigen was tested, by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, for its ability to inhibit the activity of serine proteases, i.e., trypsin, chymotrypsin and elastase. We demonstrated that the serine protease inhibitor (serpin) of SCC antigen is specific for chymotrypsin. Preincubation of chymotrypsin with recombinant SCC antigen inhibited chymotryptic digestion of gelatin and ovalbumin through the formation of sodium dodecyl sulfate‐stable complexes. These findings promote understanding of the biological functions of SCC antigen as serpin in the stratification of the normal squamous cells and in the malignant behaviour of the tumor cells.
Progress with gene‐product mapping of the Mollicutes: Mycoplasma genitalium
Authors: Wasinger Valerie C. , Cordwell Stuart J. , Cerpa‐Poljak Anne , Yan Jun X. , Gooley Andrew A. , Wilkins Marc R. , Duncan Mark W. , Harris Ray , Williams Keith L. , Humphery‐Smith Ian 
Published: May 2005

AbstractA protein map of the smallest known self‐replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two‐dimensional gel electrophoresis. A further 19 proteins were subjected to peptide‐mass fingerprinting using matrix‐assisted laser desorption ionisation‐time of flight (MALDI‐TOF) mass spectrometry and 4 were subjected to N‐terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide‐mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of ‘hierarchical’ analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or ‘proteome’ is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.
The major protein expression profile and two‐dimensional protein databse of human heart
Authors: Kovalyov Leonid I. , Shishkin Sergei S. , Efimochkin Andrei S. , Kovalyova Marina A. , Ershova Elisaveta S. , Egorov Tsezi A. , Musalyamov Alexsandr K. 
Published: May 2005

AbstractThe construction of a two‐dimensional protein database of the human heart is presented. The database contains information on about 300 abundant proteins of human myocardial tissue, including approximately 40 proteins that were identified by different methods. Each protein was characterized according to several parameters, including molecular weight, isoelectric point, name, partial sequence, subcellular localization, and genetic as well as embryonic changes.
Analysis of growth factor stimulated nucleocytoplasmic protein transport using two‐dimensional gel electrophoresis
Authors: Bedells Clare H. , Pennington Stephen R. 
Published: May 2005

AbstractThe rapid activation of gene expression by growth factors frequently involves the nucleocytoplasmic redistribution of proteins which regulate the transcription of discrete sets of genes. In particular, it has become apparent that protein kinases and transcription factors including protein kinase A, mitogen activated protein kinase, protein kinase C, NF‐χB and Stat91, can rapidly migrate from the cytosol to the nucleus in response to a wide variety of extracellular stimuli. Furthermore, it is assumed that these proteins represent important components of the signal transduction pathways which operate between the plasma membrane and the nucleus. Here two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has been used to detect proteins which undergo rapid growth factor stimulated nucleocytoplasmic redistribution. Epidermal growth factor and insulin influenced the association of 15 individual protein spots with the nucleus. Estimates of the abundance of these proteins revealed that they represented approximately 1/1000 to 1/5000 of total nuclear protein. To permit identification by microsequencing, five of the protein spots have been recovered from multiple 2‐D PAGE gels and concentrated by one‐dimensional elution‐concentration gel electrophoresis. The data obtained demonstrate that 2‐D PAGE can be used to: (i) analyse putative regulators of growth factor activated gene transcription, and (ii) purify sufficient amounts of these proteins to facilitate identification by microsequence analysis.
New types of large‐pore polyacrylamide‐agarose mixed‐bed matrices for DNA electrophoresis: Pore size estimation from Ferguson plots of DNA fragments
Authors: Chiari Marcella , D'Alesio Luca , Consonni Roberto , Righetti Pier Giorgio 
Published: May 2005

AbstractThe average pore size value of gels containing polyacrylamide, covalently linked to agarose, was found to be 30% higher than the value of a regular N,N′‐methylenebisacrylamide (Bis) cross‐linked gel of the same %T. By increasing the agarose concentration (10% of the total amount of polyacrylamide), gels containing low amounts of acrylamide (1.5–2%) are reproducibly obtained; their pore sizes are 130% larger than the pore sizes of a 4%T, 3.3%C polyacrylamide gel. In general, mixed‐bed matrices were found to be more elastic and mechanically stronger than classical polyacrylamide gels since an agarose‐induced gelation process takes place during their polymerization.
Detection of biotinylated proteins in crossed immunoelectrophoresis gels: Studies on platelet membrane receptors and microparticles
Authors: Solum Nils Olav , Holme Pål André , Pedersen Turid M. 
Published: May 2005

AbstractBiotinylation can be used as an alternative for surface labeling of cell membrane proteins. The use of the water soluble N‐hydroxysulfosuccinimide (NHSS)‐biotin or the more lipophilic N‐hydroxysuccinimide (NHS)‐biotin reagent has been investigated in the present study labeling two central receptor complexes on the platelet surface, i.e. the glycoprotein (GP) Ib–IX and the GP IIb–IIIa complexes involved in platelet adhesion and aggregation. Lack of labeling of the intracellularly located albumin was used as a negative control. The labeling has been studied using crossed immunoelectrophoresis in the PhastSystem format after extraction of the labeled cells in Triton X‐100, and it is shown that, using enzyme‐conjugated avidin and chromogenic substrates, the biotinylated proteins can be visualized directly in the dried electrophoresis gel without the need for a transfer to a blotting membrane as is used after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Suitable conditions for biotinylation and for visualization in the crossed immunoelectrophoresis gels are described. Further, surface‐biotinylation of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. For this purpose the formation and composition of the biotinylated microparticles were observed by flow cytometry and crossed immunoelectrophoresis.
Capillary electrophoresis of carboxylated carbohydrates. Part 2. Selective precolumn derivatization of sialooligosaccharides derived from gangliosides with 7‐aminonaphthalene‐1,3‐disulfonic acid fluorescing tag
Authors: Mechref Yehia , Ostrander Gary K. , Rassi Ziad El 
Published: May 2005

AbstractThe most suitable conditions for selective precolumn derivatization of sialooligosaccharides, derived from gangliosides, with 7‐aminonaphthalene‐1,3‐disulfonic acid (ANDSA) and the subsequent separation of the derivatives by capillary electrophoresis are described. ANDSA‐sialooligosaccharide derivatives, which fluoresce at 420 nm when excited at 315 nm, were readily detected in capillary electrophoresis using an on‐column lamp‐operated fluorescence detector. In addition, the precolumn derivatization described here, which exploited the reactivity of the carboxylic acid group of the sialic acid residue of the oligosaccharides, replaced each weak carboxylic acid group of the parent sugar by two strong sulfonic acid groups. This allowed for electrophoresis over a wide range of pH and improved the resolution of the derivatives when compared to those obtained with underivatized sialooligosaccharides under identical separation conditions. The separation of sialooligosaccharides was best achieved when 75 mM borate, pH 10.0, was used as the running electrolyte. The derivatization and separation conditions described herein are expected to be readily transposed to the capillary electrophoresis of other sialooligosaccharides such as those derived from glycoproteinsPresented partially in a lecture (M‐L7) at the Fourth International Symposium Workshop on Bioanalysis, Lawrence, KS, July 10–13, 1994 and as part of a lecture at Electrophoresis Forum '94, Munich, Germany, October 24–26, 1994. Part I is reference [9].
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Separation of tryptophan‐derivative enantiomers with iron‐free human serum transferrin by capillary zone electrophoresis
Authors: Kilár Ference , Fanali Salvatore 
Published: May 2005

AbstractEnantiomers can be separated by using human serum transferrin as a chiral phase. With the help of the native protein we were able to separate enantiomers with high efficiency, using a low ionic strength 2‐(N‐morpholino)ethanesulfonic acid (MES) buffer, pH 6, in capillary zone electrophoresis. Tryptophan methyl, ethyl and butyl ester enantiomers – moving towards the cathode at pH 6 – were resolved by passing through an iron‐free transferrin zone in coated capillaries. Since the isoelectric point of the iron‐free transferrin is a little higher than 6, the protein zone is either not moving in the experiment or is slowly moving towards the anode. Under the simplest experimental conditions the highest resolution was obtained for the butyl ester enantiomers and the lowest for the methyl ester ones. By changing the experimental conditions, however, this order could be reversed. The results indicate that the lengths of the alkyl chains in the enantiomers have a significant effect on the resolution, i.e., on the interaction between the protein and the separands.
Automated short tandem repeat (STR) analysis in forensic casework — a strategy for the future
Authors: Gill Peter , Kimpton Colin P. , Urquhart Andrew , Oldroyd Nicola , Millican Emma S. , Watson Stephanie K. , Downes Terry J. 
Published: May 2005

AbstractShort tandem repeat (STR) loci are routinely analysed for forensic purposes in the UK. Because small regions of DNa are amplified, successful results are more likely to be obtained from highly degraded material where the DNA fragment length may be < 500 bp. The method is superceeding conventional analysis with single locus probes (SLPs). Dimeric STR loci display stutter artefacts, hence STRs used in casework are restricted to tri or tetrameric loci. Some STRs are complex repeats and have more alleles than simple repeats – for example the locus D21S11 has 21 alleles which differ in size by 2 bp because of the presence/absence of a hexanucleotide within the block of tetrameric repeats. These loci are of great potential interest because they combine increased discriminating power with reduced potential to stutter. Multiplexing 4 different loci with different dye labelled primers (i.e. carrying out polymerase chain reaction of 4 loci simulataneously) using the ABD 373 A automated sequencer enables a large numbers of samples to be processed. In addition data aquisition and manipulation is automated so that minimum postelectrophoresis operator input is required. It is our aim to develop a system equivalent in power to that of 4 single locus probes. To achieve this we have developed an octoplex system consisting of 7 loci and a sex test (amelogenin locus) which has a probability of chance of association of 10−9; the power of this system is equivalent to that achieved by 4 conventional SLPs.
Chemiluminescent detection of DNA probes in forensic analysis
Authors: Klevan Leonard , Horton Liz , Carlson David P. , Eisenberg Arthur J. 
Published: May 2005

AbstractThe conditions for hybridization and detection of enzyme‐labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme‐linked probes which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity than observed for 32P‐labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi‐Phos® Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.
Simple protocols for typing forensic biological evidence: Chemiluminescent detection for human DNA quantitation and restriction fragment length polymorphism (RELP) analyses and manual typing of polymerase chain reaction (PCR) amplified polymorphisms
Authors: Budowle Bruce , Baechtel F. Samuel , Comey Catherine T. , Giusti Alan M. , Klevan Leonard 
Published: May 2005

AbstractMethods for identity testing are described that enable extraction of DNA from biological samples, determination of the quantity of human DNA, and genetic analyses of the materials using restriction fragment length polymorphism (RFLP) typing and/or amplified fragment length polymorphism (AMP‐FLP) typing of PCR products. The salient features of the procedures are simplicity, manual typing, nonradioactive chemiluminescent assays or silver staining for detection, and low cost. Most application‐oriented laboratories involved in forensic and/or paternity testing should be able to implement these procedures.
Mutation processes at human minisatellites
Authors: Jeffreys Alec J. , Allen Maxine J. , Armour John A. L. , Collick Andrew , Dubrova Yuri , Fretwell Neale , Guram Tara , Jobling Mark , May Celia A. , Neil David L. , Neumann Rita 
Published: May 2005

AbstractMinisatellites provide one of the most experimentally tractable systems for studying tandem repeat instability in man. Analysis of mutation processes has been greatly aided by the development of single molecule methods for recovering de novo mutants, and of techniques for exploring allele structure in detail. Application of these approaches to man has shown that minisatellites do not primarily mutate by processes such as replication slippage and unequal crossover intrinsic to the tandem repeat array. Instead, germline repeat instability is largely regulated by cis‐acting elements near the array and involves unexpectedly complex processes of gene conversion, of potential relevance to the biology of meiosis. These processes can be explored both in humans and, in principle, in transgenic mouse models of human repeat instability.
DNA fingerprinting to determine paternity in laboratory rat sperm competition experiments
Authors: Shimmin Glenn A. , Sofronidis George , Bowden Don K. , Temple‐Smith Peter D. 
Published: May 2005

AbstractPrior to this study a significant amount of research had been undertaken in the field of sperm competition in mammals. However, males of different strains have been required in each of these studies to enable paternity assignment through gene expression, which has consequently resulted in problems with differential fertilising capacity being encountered. In this study paternity assignment of progeny from sperm competition experiments with Sprague Dawley rats was achieved by multilocus DNA fingerprinting using band locus matching of individual specific banding patterns between progeny and parents. Trials with 4 restriction enzymes and 5 digoxygenin labelled probes (4 oligonucleotide and 1 cloned) achieved the highest levels of DNA fingerprint heterozygosity using AluI(CAC)5 and HinfI(CAC)5 combinations; however, paternity could not be determined in all offspring, due to a higher than expected degree of inbreeding within the rat population used in this study. This was demonstrated in subsequent comparisons of genetic diversity of three laboratory rat breeding populations from two different animal breeding facilities. Data from the rat mating study showed that, under conditions of direct sperm competition, second males given access to a mated oestrus female either 0.5 or 6.0 h after the first mating consistently required less time than the first to ejaculate: 7.6 min vs. 19.5 min (0.5 h delay); 7.8 min vs. 19.5 min (6.0 h delay). A second male siring advantage was identified using DNA fingerprinting in both delay groups for those offspring on which paternity could be determined: 0.5 h delay, 1st = 39%, 2nd = 61%; 6 h delay, 1st = 34%, 2nd = 66%.
Statistical and genetic aspects of quality control for DNA identification
Authors: Morton Newton E. , Collins Andrew 
Published: May 2005

AbstractQuality control for likelihood ratio (LR) tests on restriction fragment length polymorphisms (RFLPs) and polymerase chain reaction (PCR) markers is provided by the 4N6 program. Examples are given for tests of exclusion, coincidence, and kinship that should be used routinely in validation of DNA profiling. They confirm that “in a knowledgeable court DNA profiling is no longer exposed to risk of illogical presentation, blind acceptance or arbitrary rejection” (N. E. Morton, Eur. J. Hum. Genet. 1993, 1, 172–179). However, they remain to be implemented in national systems of quality control. There can be no effective quality control until attempts to fudge gene frequencies in order to retain the Hardy‐Weinberg assumption (recommended by the National Research Council) are abandoned in favour of the population genetic approach that takes gene frequencies from a real population and uses kinship to express genotype probabilities. Since an expert witness cannot know the identity of the culprit, he is obliged to consider multiple hypotheses without usurping the prerogative of the court to favour one of them on the basis of other evidence. It remains to be seen whether the new NRC Committee (and more importantly the international community of forensic scientists) will adopt this fundamental principle, which is especially important in populations like India with preferential endogamy and consanguineous marriages. Alternative procedures and the causes, effects, and remedies of four types of error are also considered.