bluuebunny/crossref_metadata_2025_split huggingface dataset:
Screening for urinary methadone by capillary electrophoretic immunoassays and confirmation by capillary electrophoresis‐mass spectrometry
Authors: Thormann Wolfgang , Lanz Matthias , Caslavska Jitka , Siegenthaler Peter , Portmann Rudolf 
Published: May 2005

AbstractThis paper characterizes competitive binding, electrokinetic capillary‐based immunoassays for urinary methadone using reagents which were commercialized for a fluorescence polarization immunoassay. After incubation of 25 μL urine with the reactants, a small aliquot of the mixture is applied onto a fused‐silica capillary and the unbound fluorescein‐labeled methadone tracer is monitored by capillary electrophoresis with on‐column laser‐induced fluorescence detection. Configurations in presence and absence of micelles were investigated, found to be capable of recognizing urinary methadone concentrations ⩾ 10 ng/mL, and shown to be suitable for rapid methadone screening of patient urines. Based upon shorter run times and a much better separation of free tracer and antibody‐tracer complex, conditions without micelles are preferred. For confirmation analysis of urinary methadone and its major metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), capillary electrophoresis in a pH 4.6 ammonium acetate‐acetic acid buffer was interphased to an atmospheric pressure ionization triple quadrupole mass spectrometry system. Using positive ion electrospray ionization and the tandem mass spectrometry mode with collision‐induced dissociation in the collision cell, fragmentation of the two substances was determined. For confirmation via direct urine injection or application of a urinary extract, in‐source fragmentation was employed and the first quadrupole was operated in the selected ion monitoring mode by switching between the masses of relevant precursor/product ion sets for methadone (m/z = 310, 265) and EDDP (m/z = 278, 249, 234). This capillary electrophoresis – mass spectrometry approach is shown to permit the confirmation of methadone and EDDP in patient urines that tested positive for methadone using electrokinetic capillary‐based immunoassays, a fluorescence polarization immunoassay, and capillary electrophoresis with UV absorption detection.
Capillary sample introduction of polymerase chain reaction (PCR) products separated in ultrathin slab gels
Authors: Bullard Katherine M. , Hietpas Paula Beyer , Ewing Andrew G. 
Published: May 2005

AbstractPolymerase chain reaction (PCR) amplified short tandem repeat (STR) samples from the HUMVWF locus have been analyzed using a unique sample introduction and separation technique. A single capillary is used to transfer samples onto an ultrathin slab gel (57 μm thin). This ultrathin nondenaturing polyacrylamide gel is used to separate the amplified fragments, and laser‐induced fluorescence with ethidium bromide is used for detection. The feasibility of performing STR analysis using this system has been investigated by examining the reproducibility for repeated samples. Reproducibility is examined by comparing the migration of the 14 and 17 HUMVWF alleles on three consecutive separations on the ultrathin slab gel. Using one locus, separations match in migration time with the two alleles 42s apart for each of the three consecutive separations. This technique shows potential to increase sample throughput in STR techniques although separation resolution still needs to be improved.
Sequencing using capillary electrophoresis of short tandem repeat alleles separated and purified by high performance liquid chromatography
Authors: Marino Michael A. , Devaney Joseph M. , Smith Jonathan K. , Girard James E. 
Published: May 2005

AbstractPolymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene‐divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion‐paring reverse‐phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double‐stranded DNA sequencing properties. In this study, the IP‐RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak‐captured, then cycle‐sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 μm and a total length of 47 centimeters. The total analysis time including capillary filling and pre‐electrophoresis was less than 30 min for a 220‐bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9,9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.
Myoglobin intron variation in the Gouldian Finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis
Authors: Heslewood Margaret M. , Elphinstone Martin S. , Tidemann Sonia C. , Baverstock Peter R. 
Published: May 2005

AbstractIn this study we show how the use of exon‐primed, intron‐crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine‐specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch, Erythrura gouldiae. A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low‐melting domain. Sequencing of the entire fragment from 19 Er. gouldiae revealed five nucleotide substitution differences within the low‐melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high‐melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these six alleles.
Development of standard reference materials for diagnosis of p53 mutations: Analysis by slab gel single strand conformation polymorphism
Authors: O'Connell Catherine D. , Tian Jing , Juhasz Agnes , Wenz H.‐Michael , Atha Donald H. 
Published: May 2005

AbstractWe have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5–9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base delection. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG‐SSCP). Fluorescent‐labeled PCR primers were used for amplification of specific exons for mutation detection. SG‐SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations. The separation with respect to wild type varied for each mutation examined. Subambient temperature (20°C) was preferable overall for discrimination of these mutations as a group. We intend to use this system to examine a much larger panel of p53 mutation standards that are now under development.
Elution of glycoproteins from replicas of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels
Authors: Szewczyk Boguslaw , Pilat Zbigniew , Bienkowska‐Szewczyk Krystyna , Summers Donald F. 
Published: May 2005

AbstractA method for the elution of glycoproteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) replicas of gels on polyvinylidene difluoride (PVDF) membranes is described. Ten model glycoproteins were resolved by SDS‐PAGE and then electrotransferred onto PVDF membranes. After reversible staining, glycoprotein bands were eluted with a mixture of SDS/Triton X‐100 at pH 9 or with a mixture of guanidinium hydrochloride/lysophosphatidylcholine at neutral pH. For both types of eluents, the final recoveries ranged from over 30% to about 80%. Good recoveries and mild conditions of elution render the method applicable for the structural elucidation of glycan chains.
Forensic validation of the short tandem repeat HUMACTBP2 using capillary electrophoresis
Authors: Dimo‐Simonin Noelle , Grange Fabienne , Kratzer Adelgunde , Brandt‐Casadevall Conxita , Mangin Patrice 
Published: May 2005

AbstractExperiments were performed to evaluate the forensic identification of the short tandem repeat (STR) HUMACTBP2 (human beta‐actin‐related pseudogene) using automated fluorescence‐based capillary electrophoresis. The HUMACTBP2 is a complex tetranucleotide STR locus with more than 32 alleles in the range of 202‐323 bp. The reproducibility of genetic typing using a fluorescent labeled allelic ladder was determined by comparison of the calculated fragment size after consecutive (within‐day) and nonconsecutive (day to day) injection. The maximum variation in size (window) observed for any allele was 0.23 bp for the within‐day and 0.8 bp for the day‐to‐day precision. Furthermore, it is possible to achieve a 1 bp resolution, the precision of the reproducibility assays being about 99.95%. Sixty blood samples and twenty stains were typed with both automated fluorescent sequencer ABI 373A and ABI 310. Identical genotypes were obtained with both techniques and the ABI 310 seemed to be more sensitive than the ABI 373A. A population sample of 197 unrelated individuals from southwest Switzerland was analyzed and the genotype frequencies observed were similar to those reported by others. Thirty‐one alleles and 126 genotypes were found. The observed heterozygosity was 0.934. Mixtures from two different blood samples varying in their ratio were typed and the minor fraction was detectable to about 1:10. The practical usefulness of the HUMACTBP2 is illustrated by analyzing casework samples. This validation study proves the usefulness of the HUMACTBP2 locus in forensics and the detection efficiency using fluorescent capillary electrophoresis.
Plasma protein adsorption patterns on emulsions for parenteral administration: Establishment of a protocol for two‐dimensional polyacrylamide electrophoresis
Authors: Harnisch Stephan , Müller Rainer H. 
Published: May 2005

AbstractThe two‐dimensional polyacrylamide electrophoresis (2‐D PAGE) of the plasma protein adsorption pattern previously established for polymeric nanoparticles was modified and transferred to oil in water emulsions for intravenous administration. The emulsions were incubated with citrated plasma, and separation from excess plasma was performed by centrifugation under optimized conditions: 15 000 g and three washing steps with 0.05 M phosphate buffer, pH 7.4. With this sample preparation, coalescence of droplets could be avoided and an unchanged surface area maintained, in addition the phosphate buffer minimized artificial IgG adsorption. Critical factors affecting sensitivity were contamination of the sample by oil residues and the use of thiourea in the immobilized pH gradients. Changes in the protein adsorption pattern caused by altered surface properties of the emulsion (i. e. adsorbed Poloxamer 407) were detectable when applying the optimized protocol. Knowledge of the protein adsorption patterns and their correlation to in vivo behavior opens the perspective for the development of intravenous emulsions for controlled drug delivery.
Affinity mode of capillary isoelectric focusing for the characterization of the biotin‐binding protein actinavidin
Authors: Okun Vadim M. 
Published: May 2005

AbstractDifferent modes of capillary isoelectric focusing (CIEF) with salt mobilization and zwitterionic additive to cathodic mobilizer were applied to characterize the biotin‐binding protein actinavidin and its affinity properties. The analysis is performed in a neutral coated capillary with completely eliminated electroosmotic flow. Synthetic pI standards with absorption in the visible region were used in all runs and pI determinations were based on a fitted nonlinear calibration graph. CIEF of highly purified actinavidin in native conditions was revealed in about 12 peaks in pI range 6.2–7.5, with three major forms having pI 6.80, 6.86, 6.90. CIEF of a mixture of actinavidin and increasing concentrations of two affinity ligands (biotin and biotinylated oligonucleotide) demonstrated drastic changes in the number of protein isoforms. In the latter case it resulted in only one peak (pI 5.05) when the ligand was in excess. This method, which can be named affinity CIEF, was found to be well‐suited for studying structural changes, occurring in receptor proteins upon binding with a ligand. CIEF of the protein, performed under denaturing conditions (6 M urea in a solution of carrier ampholytes) is also reported. It was revealed in three isoforms with a pI more acidic than that of native actinavidin. It is demonstrated that careful selection of denaturing conditions was necessary for the reproducible results.
Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis
Authors: Bjørnsdottir Inga , Kepp Dorte Rode , Tjørnelund Jette , Hansen Steen Honoré 
Published: May 2005

AbstractA capillary electrophoresis method for determination of the enantiomers of ibuprofen and its major phase I metabolites: 2′‐hydroxyibuprofen and 2′‐carboxyibuprofen in urine samples have been developed. Cyclodextrins and linear dextrins have been investigated as chiral selectors. Simultaneous chiral separation of the enantiomers of ibuprofen, 2′‐hydroxyibuprofen and 2′‐carboxyibuprofen was obtained using a mixture of dextrin 10 and heptakis (2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin in a 2‐[N‐morpholino]ethanesulphonic acid buffer, pH 5.26. The electroosmotic flow was reversed using hexadimethrine bromide as a buffer additive. The method can be used for the determination of the free enantiomers of ibuprofen, 2′‐hydroxyibuprofen and 2′‐carboxyibuprofen as well as for the indirect determination of their glucuronic acid conjugates in urine samples.
Microevolution during epidemic spread of Neisseria meningitidis (minireview)
Authors: Achtman Mark 
Published: May 2005

AbstractSimilar to many other naturally transformable bacteria, Neisseria meningitidis has yielded many examples where horizontal genetic exchange has resulted in genetic variation of individual strains. Epidemic strains are purified of genetic variants due to bottlenecks during the spread from country to country, resulting in clonal descent. Occasionally, clonal replacement also occurs during epidemic spread. These processes occur rapidly in serogroup A meningococci; such that after their descent from a common ancestor, clonally related bacteria have diversified at numerous loci within the last decades.
Mosaic genes and their role in penicillin‐resistant Streptococcus pneumoniae (minireview)
Authors: Hakenbeck Regine 
Published: May 2005

AbstractPenicillin resistance in clinical isolates of Streptococcus pneumoniae is mediated by mosaic genes encoding altered penicillin binding proteins. Mosaic sequence blocks are the result of a genetic exchange between related streptococcal species. It is likely that resistance has emerged in commensal streptococci before being transferred into the pneumococcus. Closely related mosaic genes are found in different pneumococcal clones and in different streptococcal species isolated worldwide since the first reports on such strains in the late 70s, demonstrating the importance of commensal streptococci for the spread of selectable markers in naturally transforming pathogens.
Activity staining of pectinesterase on polyacrylamide gels after acidic or sodium dodecyl sulfate electrophoresis
Authors: Hou Wen‐Chi , Lin Yaw‐Huei 
Published: May 2005

AbstractPectinesterase (PE), from commercial orange peels or ammonium sulfate fractionation (50–80% saturation) of pea pods, was detected on polyacrylamide gels after native acidic polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)‐PAGE by using the synthetic substrate β‐naphthyl acetate (β‐NA). The release of β‐naphthol (at 322 nm) from β‐NA was proportional to PE activity. The PE activity bands on polyacrylamide gels after native acidic PAGE or SDS‐PAGE were stained with a combination of tetrazotized o‐dianisidine and β‐NA. This fast and sensitive method can be used for enzyme purification and characterization.
Use of thiourea to increase the solubility of membrane proteins in two‐dimensional electrophoresis
Authors: Rabilloud Thierry 
Published: May 2005

AbstractThe separation of membrane proteins by high‐resolution two‐dimensional electrophoresis was carried out. At high loads, these proteins are prone to precipitation, resulting in poor resolution. It is shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing. As thiourea inhibits acrylamide polymerization, a modified photopolymerization system must be used. These modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two‐dimensional gels.
A two‐dimensional electrophoretic study of serum amyloid A and C‐reactive protein in infants and children
Authors: Bruun Cathrine Foyn , Sanchez Jean‐Charles , Hochstrasser Denis F. , Marhaug Gudmund , Husby Gunnar 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to analyze C‐reactive – (CRP) and serum amyloid A protein (SAA) in infants and children. Five SAA isotypes were identified. CRP showed vertical streaking, and its optical density values correlated with immunoturbidimetric measurements. As evaluated by densitometry, both proteins showed an age‐dependent variation. In more than 50% of the neonates, SAA was present in equal or higher amounts than CRP, and only SAA1α could be detected. In children, CRP was expressed in higher amounts than SAA, and both SAA1α and SAA2α were present. N‐terminally modified forms of both isotypes were present regardless of age, including in premature infants. These results suggest that the overall synthesis of the gene products SAA1α and SAA2α is developmentally regulated, but at the same time that their N‐terminal processing occurs independently of developmental factors. The presented data suggest that SAA has an important function in neonates, and that the role of SAA as an infection marker in this population should be investigated further.
Divergent and common groups of proteins in glands of venomous snakes
Authors: Rioux Vincent , Gerbod Marie‐Christine , Bouet Françoise , Ménez André , Galat Andrzej 
Published: May 2005

AbstractProtein contents of venom‐producing glands from the sea‐snake Laticauda colubrina (LC) and terrestrial Vipera Russelli (VR) were studied using high‐resolution two‐dimensional gels: isoelectric focusing followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (IEF/SDS‐PAGE) and nonequilibrium pH gradient electrophoresis (NEPHGE) followed by SDS‐PAGE. Tentative identities of numerous proteins were established using their amino acid compositions and in certain cases the identities were verified by microsequencing of their N‐terminals and internal fragments. As expected, we found several proteins known to be present in the venom of the respective snakes. These include numerous isoforms of phospholipase A2 (PLA2) in both snake glands, various neurotoxins in LC glands and factor IX/factor X‐binding protein, hemorrhagic factor and coagulation factor X activating enzyme in Russell's viper glands (VR). Not unexpectedly, we also found a number of cell housekeeping proteins, cytoskeletal proteins, proteins that are necessary for folding, such as heat‐shock proteins, protein disulfide‐isomerase and peptidylprolyl cis/trans isomerases. Unexpectedly, however, the glands of Laticauda colubrina and Russell's viper include a large quantity of antihemmorhagic factor and inhibitor of PLA2, respectively, that have been previously described in snake plasma. The possible reason associated with the presence of these components in venom glands is discussed.
Two‐dimensional gel electrophoresis separation and N‐terminal sequence analysis of proteins from Clostridium pasteurianum W5
Authors: Flengsrud Ragnar , Skjeldal Lars 
Published: May 2005

AbstractProteins from Clostridium pasteurianum were separated by two‐dimensional gel electrophoresis. Amino‐terminal sequence determination and sequence analysis allowed the identification of 20 proteins, while 11 protein sequences remained unidentified and one protein appeared to have a blocked amino terminus.
Two‐dimensional gel database of human breast carcinoma cell expressed proteins: An update
Authors: Rasmussen Richele K. , Ji Hong , Eddes James S. , Zugaro Lisa M. , Reid Gavin E. , Simpson Richard J. , Dorow Donna S. 
Published: May 2005

AbstractPreviously, we reported a two‐dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA‐MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588–598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X‐114 solubilised membrane proteins from MDA‐MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X‐114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X‐114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS‐polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric‐based procedure for identifying two‐dimensional electrophoresis (2‐DE) gel‐resolved proteins, combining in‐gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed‐phase high‐performance liquid chromatography (RP‐HPLC) peptide mapping and electrospray ionisation — ion trap — mass spectrometry.