bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Minisatellites 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

Prior 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

Quality 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.
Polymerase chain reaction‐based assays for the characterisation of plant genetic resources
Authors: Powell Wayne , Orozco‐Castillo Carlos , Chalmers Kenneth J. , Provan Jim , Waugh Robbie
Published: May 2005

Plant genetic resources are an important component of biodiversity and provide the basic genetic variability that allow new and improved cultivars to be developed. Numerous germplasm collections have been established and it is important to establish that such collections are representative and accessible to breeders and biotechnologists. Molecular markers provide the best estimate of genetic diversity since they are independent of the confounding effects of environmental factors. Assays based on the polymerase chain reaction (PCR) are considered to meet both the technical and genetical requirements for the characterisation of plant and animal genetic resources. Two main approaches are described, based on anonymous and defined primers. The use of both randomly amplified polymorphic DNA (RAPD) and microsatellites or simple sequence repeats (SSR) for the characterisation of perennial tree species, and distribution of variability within gene pools is reported. The detection of interspecific gene introgression between coffee species with RAPD markers is described together with the use of microsatellites to genotype potato. The use of PCR‐based assays will facilitate the evaluation and utilisation of plant genetic resources.
DNA profiling of banana and plantain cultivars using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers
Authors: Bhat K. Venkataramana , Jarret Robert L. , Rana Rai Singh
Published: May 2005

Polymerase chain reaction (PCR) amplification of genomic DNA from 57 Musa cultivars with 60 random 10‐mer primers generated 605 polymorphic amplification products which were useful in unambiguous cultivar identifications. Unweighted pair‐group method analysis of this data grouped the cultivars into specific clusters depending on their genomic similarities. The diploid ancestral species of cultivated banana and plantains, namely Musa acuminata ssp malaccensis, an A genome donor and M. balbisiana, a B genome donor, were farthest apart from each other in the phenogram. The edible fruit yielding cultivars with the genomic constitutions AA, AAA, AB, AAB, ABB and ABBB grouped in different clusters according to overall genetic homologies. The restriction fragment length polymorphisms (RFLPs) prevalent among the cultivars were studied by hybridizations of 19 random genomic clones to blots of HindIII, EcoRI and MspI digests. Cluster analysis of these data on 107 polymorphic alleles resulted in a phenogram comparable to the one obtained with random amplified polymorphic DNA (RAPD) analysis. Two multilocus probes useful in distinguishing all the 57 cultivars analyzed were also identified. The A and B types of cytoplasms in the cultivars were further distinguished by hybridization of heterologous chloroplast DNA probes. Results showed that use of different kinds of molecular markers in gene banks is essential for characterization and classification of germplasm collections.
Protein variability in male and female Wistar rat liver proteins
Authors: Steiner Sandra , Wahl Daniel , del Carmen Varela Maria , Aicher Lothar , Prieto Pilar
Published: May 2005

In the emerging field of mechanistic toxicology, growing attention is being paid to the interpretation of sex‐related toxic responses. The Wistar rat outbred stock is a frequently used rodent for toxicity testing. Outbred strains, which display a relatively high degree of genetic variability between the individual animals are often used in risk assessment, as they are considered to best approximate the variability present in a human population. In this study two‐dimensional (2‐D) protein gel electrophoresis was applied to investigate the liver protein patterns of male and female Wistar rats and to search for (i) sex‐related and (ii) interindividual qualitative and quantitative differences in protein expression. Among the sexes, six proteins were detected that were shown to be exclusively present in male rats and one that was present only in females. A male‐specific protein was tentatively assigned to α2u globulin. Seven protein spots showed statistically significant abundance changes (p < 0.001) between males and females, one was tentatively assigned to heme oxygenase 1 and another to the 23 kDa morphin‐binding protein. Four sets of protein spots were detected that showed positional shifts in the individual patterns and are likely to represent polymorphic proteins inherent in the Wistar rat. These results form a valuable basis for future investigations of drug‐induced changes in the male and female Wistar rat liver pattern.
Sample self‐stacking and sample stacking in zone electrophoresis with major sample components of like charge: General model and scheme of possible modes
Authors: Gebauer Petr , Thormann Wolfgang , Boček Petr
Published: May 2005

A theoretical study is presented of zone electrophoretic behavior of samples that contain one or more minor analytes and at least one major ionogenic component of like charge. Based on a simple model comprising weak univalent anionic electrolytes, conditions are derived under which analytes are temporarily focused isotachophoretically into very narrow zones by a sample self‐stacking effect provided by the major sample components. Requirements for minimal/maximal mobility and a background coion concentration dependent minimal concentration of a major sample component (stacker) are presented. For systems in which sample self‐stacking does not apply, an expression for the concentrating factor is derived that involves the effects of both nonselective (classical) and selective sample stacking, the latter being a consequence of electrophoretic separation of the minor analyte from the major component. The theory derived is discussed with selected model examples by using both numerical calculation and computer simulation.
Demethylation of a repetitive DNA sequence in human cancers
Authors: Miwa Wataru , Yashima Kazuo , Sekine Teruaki , Sekiya Takao
Published: May 2005

To detect DNA alterations in unknown regions in human cancers, we have performed restriction landmark genomic scanning (RLGS) analysis of DNA isolated from cancer and normal cells. One spot with a highly intensified signal was detected in DNA from all six malignant melanoma cell lines, two of five colon cancer cell lines and one of six pancreatic cancer cell lines analyzed. In DNA from normal cells, two placentas and seven cultured lymphocytes, the signal of this spot was not intense. The DNA fragment corresponding to the spot was cloned. By nucleotide sequence analysis, the DNA fragment was revealed to be a part of a repeating unit of a 13 kbp nucleotide sequence of which 200 copies were located in chromosome 8q21. Southern blotting analysis using the cloned fragment as a probe demonstrated that the intensified signal for the DNA fragment observed in cancer cells was due to demethylation in the recognition sequence of the NotI restriction enzyme. The results suggest that marked demethylation in the repeating units might be associated with the genesis or progression of some types of cancers.
Application of fluorescence in situ hybridization in genome analysis of the mouse
Authors: Matsuda Yoichi , Chapman Verne M.
Published: May 2005

Fluorescence in situ hybridization (FISH) is an effective technique for localizing cloned DNA probes directly onto metaphase chromosomes. Human genome mapping using FISH has been significantly enhanced by the development of new techniques, especially high‐resolution gene mapping with direct R‐banding FISH and physical gene ordering with multi‐color FISH. By contrast, FISH techniques have not been put to practical‐use for the analysis of the mouse genome compared with the human. We have developed and modified FISH techniques for use in mouse genome analysis. In this article we summarize and review our recent results with FISH analyses in the following studies: (i) high‐resolution gene mapping with the direct R‐banding FISH, (ii) analysis of chromosomal rearrangement with multi‐color FISH, (iii) establishment of centromere mapping with the major satellite DNA probe (iv) analysis of chromatin structure in meiotic cells, and (v) application of FISH in cytogenetic studies of genetic variation in the mouse, showing that these applications of FISH are very useful for mouse genome analysis.
Genomic mismatch scanning: Current progress and potential applications
Authors: Nelson Stanley F.
Published: May 2005

Genomic mismatch scanning (GMS) is a new method of genetic mapping which attempts to purify and map the regions of identity between two complex genomes in a single test. Identical DNA fragments from two genomic sources are enriched in two steps: (i) after reannealing of the two genomes, heterohybrids are purified by using a combination of a restriction methylase and methylation‐sensitive endonucleases, (ii) heterohybrids that contain mismatches are nicked in vitro by the E. coli MutHLS mismatch repair system and are eliminated subsequently from the pool, leaving only mismatch‐free heterohybrids. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step onto metaphase chromosomes or ordered DNA arrays. The principal advantages of GMS are (i) it approaches the theoretical limit of mapping power and resolution offered by an arbitrarily dense set of completely informative polymorphic markers and (ii) it results in a great increase in the effective number of informative markers without a corresponding increase in the number of individual tests. Thus, it should provide an efficient method for affected‐relative‐pair linkage mapping and for linkage disequilibrium mapping. In addition, a variation of GMS may allow rapid genomic scanning for regions of homozygosity‐by‐descent or somatic loss‐of‐heterozygosity. The feasibility of GMS has been validated in the 15 mb genome of Saccharomyces cerevisiae. This article discusses the principles of GMS, the application to more complex genomes, and the possible uses of GMS.
Rapid and effective Western blotting of histones from acid‐urea‐Triton and sodium docecyl sulfate polyacrylamide gels: Two different approaches depending on the subsequent qualitative or quantitative analysis
Authors: Thiriet Christophe , Albert Philippe
Published: May 2005

An improved method for the electrophoretic transfer of histones from sodium dodecyl sulfate (SDS) and acetic acid‐urea‐Triton X‐100 (AUT) polyacrylamide gels onto nitrocellulose membranes is described. In the case of SDS‐gels, it was not essential to equilibrate them before transfer while for the AUT‐gels, an equilibration step is essential to prevent the interference of Triton X‐100 with the binding of histones to nitrocellulose. Transfer efficiency was different for different histone classes. Two procedures were developed: (i) one suitable for qualitative studies, and (ii) another for quantitative transfer.
Separation and detection of amino acids and their enantiomers by capillary electrophoresis: A review
Authors: Issaq Haleem J. , Chan King C.
Published: May 2005

Since its introduction as an analytical technique capillary electrophoresis has been used for the separation of amino acids and their enantiomers; over 150 studies have been published to date. This review deals with their separation and detection. Amino acids have been resolved using both capillary zone electrophoresis and micellar electrokinetic chromatography. Pre‐column derivatization schemes which are employed for the sensitive detection of amino acids are discussed. Criteria for the selection of the pre‐ or post‐column derivatizing agent, chromophore or fluorophore, are presented. Detection systems, direct and indirect, that have been used are given with emphasis on fluorogenic reagents and laser induced fluorescence detection. Also, procedures for the separation of amino acid enantiomers are discussed and illustrated.
Enantiomeric separation of amino acids using micellar electrokinetic chromatography after pre‐column derivatization with the chiral reagent 1‐(9‐fluorenyl)‐ethyl chloroformate
Authors: Chan King C. , Muschik Gary M. , Issaq Haleem J.
Published: May 2005

Direct enantiomeric separations of some racemic amino acids derivatized with 9‐fluorenylmethyl chloroformate were obtained using cyclodextrin‐modified micellar electrokinetic chromatography (CD/MEKC) with a buffer made up of 5 mM sodium borate (pH 9.2), 150 mM sodium dodecyl sulfate (SDS) and 40 mM γ‐CD. Alternatively, enantiomeric separations were also achieved indirectly using MEKC after pre‐column derivatization with (+)‐1‐(9‐fluorenyl) ethyl chloroformate (FLEC). Using either a 10 mM sodium phosphate (pH 6.8) or a 5 mM sodium borate buffer (pH 9.2), each of which contained 25 mM SDS and 10–15% of acetonitrile, FLEC‐derivatized serine, alanine, valine, methionine, leucine, phenylalanine, tryptophan, and their diastereomeric pairs were all separated: the L‐isomers migrated faster than the corresponding D‐isomers. However, when (−)‐FLEC was used for derivatization, the D‐isomers migrated faster than the corresponding L‐isomers. Also, the diastereomers of aspartic acid, glutamic acid, and proline were resolved using a 10 mM sodium citrate buffer (pH 4.4). Using KrF (248 nm) laser‐induced fluorescence, the detection limit of (+)‐FLEC derivatized DL‐amino acids was obtained at the nM level, which was about 100 × more sensitive than UV absorption at 200 nm. Analyte concentrations as low as 3 × 10−8 M (DL‐Val) could be derivatized with (+)‐FLEC.
Application of capillary electrophoresis to amino acid sequencing of peptide
Authors: Kim Nam Jung , Kim Jung Ho , Lee Kong‐Joo
Published: May 2005

Separations of twenty phenylthiohydantoin (PTH) amino acids, and amino acid derivatives, resulting from the Edman degradation of peptides and proteins, were optimized for peptide sequencing by capillary electrophoresis. Manual sequencing of angiotensin II was performed by Edman degradation and capillary electrophoresis of the PTH amino acid obtained after each cycle. The results were compared with those of an automated conventional protein sequencer. Interfacing capillary electrophoresis with Edman degradation provides an additional option for protein sequencing.
Fast, high‐resolution (capillary) electrophoresis in buffers designed for high field strengths
Authors: Hjertén Stellan , Valtcheva Ludmila , Elenbring Karin , Liao Jia‐Li
Published: May 2005

Capillary electrophoresis in conventional buffers and in 50 μ capillaries permits field strengths as high as 300–500 V/cm with acceptably low thermal zone deformation. However, still higher field strengths (up to at least 2000 V/cm) can be applied without a decrease in resolution if the experiments are performed in the buffers described in this paper. Characteristic of these buffers is their low electrical conductivity and yet satisfactory buffering capacity accomplished either (i) by selecting buffer constituents of relatively high molecular weight and small net charge or (ii) by fractionation of carrier ampholytes (originally introduced for isoelectric focusing experiments) into a series of narrow pH range fractions and using these fractions as buffers, or (iii) by selecting an ampholyte with two acidic groups and one basic group (or one acidic group and two basic groups) and with a pI value close to two of its pK values. In such buffers, aromatic carboxylic acids and proteins used as model substances could be analyzed rapidly. For instance, albumin and transferrin were separated at 30000 V (1.99 μA) in 15 cm long fused silica capillaries (50 μ ID) within 40 s and the carboxylic acids within 25 s. The resolution was similar to that obtained at standard voltage (5000 V; 0.33 μA), but the analysis time was reduced sixfold. Although not verified experimentally we also suggest the use of relatively high‐molecular‐weight polyoxyethylene derivatized with one acidic group (for instance, boric acid) and one basic group (an amine), both having the same pK value, which should afford both a very high buffering capacity and very low electrical conductivity (at low buffer concentrations).
Effect of buffer composition on the migration order and separation of histone H1 subtypes
Authors: Lindner Herbert , Helliger Wilfried , Sarg Bettina , Meraner Christoph
Published: May 2005

The effects of different buffer concentrations and compositions on the elution order and separation of H1 histone subtypes and their phosphorylated modifications isolated from several species was studied using high‐performance capillary electrophoresis (CE). Various cations and anions were tested in an untreated silica capillary and low pH buffers, in the presence of the dynamic coating agent hydroxypropylmethyl cellulose. It was found that the cations and anions of buffers have a remarkable influence on both the efficiency and the selectivity of protein separations. A triethylammonium methanephosphonate system proved efficacious for the separation of rat histone subtype H1c from H1e and a perchlorate/triethylammonium phosphate system for the analysis of chicken and mouse linker histones. CE provides an attractive alternative to high‐performance liquid chromatography and conventional gel electrophoresis.
Ribbon channel plate rotating drum DNA sequencing device
Authors: Douthart Richard J. , Welt Marc , Walling Leslie
Published: May 2005

A new design DNA sequencing electrophoresis device is described. The device, called the ribbon channeled plate rotating drum (rprd), consists of two major components, the plate assembly and the drum assembly. The plate assembly contains a machined or etched plate of individual micro‐channels called the ribbon channeled plate. The ribbon channeled plate and other components of the plate assembly combine the advantages of thin gels and capillary arrays in a single unit with few of the disadvantages. The other major component of rprd is the drum assembly, which facilitates direct blotting onto deposition membranes affixed to a large plastic drum. The drum with attached membrane and deposited electrophoretically resolved ladders is easily moved to special units facilitating downstream processing and detection. The drum unit, although versatile, is specifically designed to be used with multiplex sequencing.
The use of a nonradioactively labelled probe system in an electrophoretic ribotyping method for the differentiation of strains of coagulase‐negative staphylococci
Authors: Hawcroft David M. , Geary Colin
Published: May 2005

Bacteria of the genus Staphylococcus are notoriously difficult to classify and identify; many routine hospital laboratories use only simple, older techniques to segregate them into broad groups. Recent studies have demonstrated that subspecific strains vary in their clinical significance and are of increasing importance in hospital‐derived infections; this has led to a need for better methods of classification and identification. Restriction fragment length polymorphism analysis of genes for ribosomal RNA (‘ribotyping’) is potentially very useful since these genes occur in multiple copies in the genome, and are relatively stable. The restriction fragment patterns are sufficiently different to be usable in classification and identification. This paper considers an electrophoretic method for the separation of these restriction gragments which has the advantages of using a nonradioactive probe and a simple colour signal generating system for its evaluation. The technical principles are universal and would allow the procedure to be developed for other groups of microorganisms.
Glycoproteins in human parotid saliva assessed by lectin probes after resolution by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis
Authors: Carpenter Guy H. , Proctor Gordon B. , Pankhurst Caroline L. , Linden Roger W. , Shori Deepak K. , Zhang Xiao S.
Published: May 2005

Human parotid salivary glycoproteins separated by gradient sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electroblotted onto nitrocellulose have been investigated using a battery of biotinylated lectin probes of characterized sugar specificity. Lectin binding, detected on blots using avidin‐biotin complex (ABC) and a chemiluminescence generating substrate, was recorded on photographic film and compared with the original fluorescein isothiocyanate (FITC) stained blots or with Coomassie Brilliant Blue R‐250‐stained gels run in parallel. A number of glycoprotein bands which were undetected by protein stains or the periodic acid Schiff reaction were revealed by lectins. Binding by lectins from Concanavalia ensiformis, Lens culinaris, Limax flavus, Phaseolus vulgaris, Ricinus communis, Triticum vulgaris, Lotus tetragonobulus and Ulex europaeus indicated that sialylated and fucosylated triantennary and bisected, N‐linked complex sugar chains were present on many glycoproteins in addition to the major glycosylated proline‐rich glycoprotein (GI). Binding with lectins from Arachis hypogaea and Dolichos biflorus indicated that the O‐linked sugar chains were confined to the α‐heavy chain of Ig A. Comparison of lectin binding in samples from five healthy individuals revealed differences in a number of flycoproteins in addition to the previously characterized GI and CON 1/CON 2 polymorphisms and demonstrated that the H blood group antigen was expressed mainly on GI in parotid saliva. This study will be used as a basis upon which to study salivary glycoproteins in diseases affecting parotid glands.
Analysis of heparin‐like pharmaceuticals by capillary zone electrophoresis and isotachophoresis
Authors: Malá Zdeňka , Křváková Ludmila , Boček Petr
Published: May 2005

Synthetic sulfated bis‐lactobionic acid amides are important heparin‐like pharmaceuticals. The synthesis of these compounds yields molecules differing in the number of sulfate groups, and during the isolation procedure, the required species may partially decompose or take in some impurities. This article shows that capillary zone electrophoresis may serve well as an expedient method for the analysis of the above‐mentioned pharmaceuticals. Complex‐forming equilibria between the analytes and bivalent cations present in the background electrolyte bring selectivity necessary for the separation, and the detection at various wavelengths serves as an aid in the characterization of admixtures, decomposition products, and impurities. Capillary isotachophoresis may also be used for the analysis of these species, bringing about the potential of micropreparation of individual compounds and opening the chance for continuous free‐flow electrophoresis.
Dextrin sulfopropyl ether: A novel anionic chiral buffer additive for enantiomer separation by electrokinetic chromatography
Authors: Jung Martin , Börnsen K. Olaf , Francotte Eric
Published: May 2005

Dextrin 10 sulfopropyl ether (DSPE) was prepared and characterized matrix‐assisted laser‐desorption ionization‐mass spectrometry (MALDI‐M) using a recently developed matrix. The anionic compound was used as a novel chiral buffer additive for enantiomer separation by capillary electrophoresis, thereby changing into the elektrokinetic chromatography (EKC) mode. DSPE was systematically evaluated as a chiral selector and was compared to the respective nonderivatized maltodextrin. DSPE showed an increased separation power for cationic racemic solutes. Although not quite as versatile and powerful as cyclodextrins, the inexpensive dextrin 10 and its derivative DSPE showed remarkable enantioselectivity in some cases.
Separation of DNA sequencing fragments using an automated capillary electrophoresis instrument
Authors: Karger Achim E.
Published: May 2005

Rapid, high‐resolution separation of DNA sequencing fragments by capillary gel electrophoresis using an automated, commercially available instrument is presented. The effect of column lengths and electric field strength on the resolution of sequencing fragments as well as the sensitivity of laser‐induced rescence (LIF) detection was investigated. Using a short capillary of 20 length, which results in a U‐shape of the capillary in the capillary cartridge very high separation efficiency, up to 17 × 106 theoretical plates per m, is obtained. Analysis of the band broadening factors revealed that the resolution on the short column is predominantly determined by axial diffusion and to a minor extent by detection zone width. Presumably due to the coiling of longer capillaries in the capillary cartridge, increasing the capillary length does increase the separation efficiency as predicted for diffusion‐limited separation. The concentration limit to detection (signal‐to‐noise ratio = 2) is 0.2 × 10−1 M of fluorescein‐labeled oligonucleotide primer under the separating conditions for DNA sequencing samples. Increasing the electric field strength from 100 to 175 V/cm improved resolution and at the same time approximately doubled the sequencing speed. Fragments up to 500 nucleotides in length are resolved in less than 50 min.
Capillary zone electrophoresis and micellar electrokinetic chromatography, with taurodeoxycholate as micellar agent, of protein kinase A peptide substrates
Authors: Beijersten Ingegerd , Westerlund Douglas
Published: May 2005

The separation of protein kinase A peptide substrates with the general formula ‐X‐Arg‐Arg‐Ala‐Ser‐Y‐, where X and Y may be the same or different amino aicds, was studied by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Taurodeoxycholate (TDC) was used as the micellar agent. CZE was effective in separating a peptide series differing in the number of amino acids, but not for a series with a difference in the terminating amino acid. For the latter series, MEKC generally gave a higher selectivity, but some of the peptide pairs were more easily separated by CZE, demonstrating the complementary character of the two techniques. The efficiency of the MEKC system was typically < 50% of that of CZE, but its higher selectivity generally outbalanced the lower efficiency regarding resolution. The distribution of the peptides to the micelles was studied by determination of retention factors. Electrostatic and hydrophobic forces were found to be determining factors in the distribution; the most highly charged basic peptides were most heavily distributed, and for peptides with the same charge those containing more hydrophobic amino acids were more strongly distributed. The contribution of some structural features to the distribution degree was also determined.
High resolution density gradient electrophoresis of cellular organelles
Authors: Tulp Abraham , Verwoerd Desirée , Fernandez‐Borja Mar , Neefjes Jacques , Hart Augustinus A. M.
Published: May 2005

A density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 × 2.2 cm) containing a 0–5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O‐ring. The top circular platium anode allows for upward electrophoresis (80–100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle‐enclosed enzymes.
Chemiluminescent enhanced CD47 detection on Western blotting
Authors: Yuan Fang Fang , Mihrshahi Seema , Fletcher Anne
Published: May 2005

CD47 is a leucocyte antigen of wide tissue distribution including platelets and erythrocytes. It has been found that anti‐CD47 antibodies may cause platelet activation. Sensitive techniques are therefore needed for biochemical investigation of platelet glycoproteins including CD47. Conventional Western blotting techniques using horseradish peroxidase‐conjugated antibody and 3,3′‐diaminobenzidine (DAB) substrate resulted in unsatisfactory results with CD47 due to low sensitivity. When chemiluminescent detection with horseradish peroxidase‐conjugated antibody was used in conjunction with Western blotting, detection of the CD47 molecule was greatly enhanced. A protein of 47–55 kDa was immunoblotted from platelets by anti‐CD47 while the mAb immunoblotted a broader band (45–60 kDa) from erythrocyte membranes. Thus, there may be different forms of CD47 present on platelets and erythrocytes. The results indicate that Western blotting with the chemiluminescent detection technique is a useful and sensitive alternative for the study of human platelet antigens.
Analysis of recombinant and native CD4 by one‐ and two‐dimensional gel electrophoresis
Authors: Lynch Garry W. , Dearden Mark , Sloane Andrew J. , Humphery‐Smith Ian , Cunningham Anthony L.
Published: May 2005

Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one‐ and two‐dimensional (2‐DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of ∼ 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4°C) of cell‐(CEM‐T4, THP‐1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS‐PAGE immunoblotting, using anti‐CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full‐length native CD4 from lymphoid MT2 and CEM‐T4 cell extracts, however, revealed both monomeric and higher‐order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1‐DE and 2‐DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.
Different electrophoretic techniques produce conflicting data in the analysis of myocardial samples from dilated cardiomyopathy patients: Protein levels do not necessarily reflect mRNA levels
Authors: Dos Remedios Cristobal G. , Berry Desirée A. , Carter Laura K. , Coumans Joëlle V. F. , Heinke Monique Y. , Kiessling Peter C. , Seeto Reginald K. , Thorvaldson Tom , Trahair Toby , Yeoh Thomas , Yao Mu , Gunning Peter W. , Hardeman Edna , Humphery‐Smith Ian , Naidoo Daya , Keogh Anne
Published: May 2005

A variety of electrophoretic techniques were used to search for potential causes of human dilated cardiomyopathy (DCM). Northern blots were used to quantify α‐cardiac and α‐skeletal muscle actins, and β‐myosin heavy chain mRNAs which are the predominant expressed isoform species. We found a wide range of mRNA levels expressed in both DCM and nondiseased (ND) samples of left ventricles. However, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels of the same heart samples revealed a stable and constant ratio of actin and myosin. Dystrophin deficiency might account for the DCM symptoms and so dystrophin levels of DCM and ND samples were evaluated using Western blots probed with monoclonal antibodies for the N‐, C‐ and mid‐rod portions of this protein. We found that dystrophin levels were constant in all 29 DCM and 5 ND samples suggesting that dystrophin deficiency is probably not a contributing cause. We explored the possibility that terminal failure may be due to an apoptotic‐like event in the cardiomyocytes. Zymograms of DCM and ND samples revealed a significant increase in DNase I activity in the DCM group compared to the ND samples. These data raise the possibility that end‐stage failure may be associated with apoptosis.
Genetic screening and testing by induced heteroduplex formation
Authors: Wood Nigel , Bidwell Jeffrey
Published: May 2005

Clustered point mutations and small deletions or insertions within DNA are amenable to rapid analysis using induced heteroduplex formation. A single synthetic molecule (universal heteroduplex generator, UHG) may detect any of a series of such mutations following amplification by the polymerase chain reaction. This paper illustrates the use of UHG‐based DNA heteroduplex analysis by describing the construction, properties, and methods of use of five UHGs, designed for genetic screening and testing of the inherited metabolic diseases: phenylketonuria, sickle‐cell disease, cystic fibrosis, von Willebrand's disease type 2B, and mamman‐binding lectin deficiency. In all cases, identification of multiple disease‐associated genotypes is possible using a single UHG.
Capillary electrophoresis with pulsed amperometric detection of carbohydrates and glycopeptides
Authors: Weber Paul L. , Lunte Susan M.
Published: May 2005

The utility of capillary electrophoresis with pulsed amperometric detection (CE‐PAD) for the analysis of carbohydrate‐containing samples from a variety of biological sources is described. CE‐PAD was used to separate a mixture of oligosaccharides obtained from bovine fetuin and to monitor the desialylation process used in the characterization of the oligosaccharides. Additionally, the high resolving power of the system was demonstrated using a series of glycopeptides obtained from recombinant coagulation factor VIIa, which possess the same decapeptide core but differ in the extent of sialylation. Deglycosylation of these glycopeptides for characterization purposes resulted in a mixture of carbohydrates and peptides. Unlike CE with UV detection, this system gave good responses for all analytes, demonstrating the unique ability of PAD to respond to the electrochemical features of diverse classes of biomolecules such as carbohydrates and peptides. Finally, CE‐PAD was applied to the analysis of a tryptic digest of recombinant tissue plasminogen activator. The use of different detection potentials in sequential runs on a sample gave structural information about the peptides, such as glycosylation. A brief review of prior applications of CE‐PAD to the analysis of standard mixtures of simple saccharides is also presented.
Separation of 8‐aminonaphthalene‐1,3,6‐trisulfonate (ANTS)‐labeled oligomers containing galacturonic acid by capillary electrophoresis: Application to determining the substrate specificity of endopolygalacturonases
Authors: Mort Andrew J. , Chen Emily Min Wu
Published: May 2005

Slight modifications of published procedures (Stefansson, M. and Novotny, M., Carbohydr. Res. 1994, 258 1–9) allow separation by relative size of 8‐aminonaphthalene‐1,3,6‐trisulfonate (ANTS)‐labeled neutral and acidic oligosaccharides by electrophoresis in uncoated capillaries. The separations are performed at pH 2.5 to suppress both any charge from carboxylic acid groups on the oligosaccharides and electroosmotic flow. Utility of the procedure is demonstrated by its application to characterization of substrate specificity of endopolygalacturonases. The results show that both a fungal and a bacterial endopolygalacturonase need four adjacent nonesterified galacturonic acid residues in a pectin to be able to act.
High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides
Authors: Karamanos Nikos K. , Vanky Peter , Tzanakakis George N. , Hjerpe Anders
Published: May 2005

A rapid, sensitive and accurate high‐performance capillary electrophoresis method is described for the determination of the sulfation pattern of heparin and heparan sulfate disaccharides. The analysis, performed after enzymic degradation of the polysaccharides with heparinase and heparinases II and III in combination, yields highly UV‐absorbing Δ‐disaccharides. The separation is performed with reversed polarity using 15 mM phosphate buffer, pH 3.50. This method is superior to others since all known 12 disaccharides carrying N‐acetylated, N‐sulfated or unsubstituent glucosamine can be separated in a single run of 15 min. At the highest sensitivity the analysis consumes only a few femtograms of glycosaminoglycan and allows a determination of Δ‐disaccharides at the attomole level.
Profiling of oligosaccharide‐mediated microheterogeneity of a monoclonal antibody by capillary electrophoresis
Authors: Hoffstetter‐Kuhn Sabrina , Alt Gerlinde , Kuhn Reinhard
Published: May 2005

Based on complex formation of borate with carbohydrates in alkaline solutions, the oligosaccharide microheterogeneity of a monoclonal antibody was studied using capillary zone electrophoresis. In borate buffers characteristic separation patterns were found that could be attributed to the same antibody by their UV spectra, while in a phosphate buffer, under otherwise the same conditions, only a single peak was observed. N‐ and O‐glycans were chemically hydrolyzed by trifluoromethane sulfonic acid, resulting in a completely deglycosylated protein; alternatively, N‐glycans were enzymatically cleaved by incubation with peptide N‐glycosidase F (PNGase F). In both approaches a changed antibody pattern was detected, indicating that the separation is due to carbohydrate microheterogeneity of the protein. Deglycosylation of the antibody by treatment with PNGase F was investigated by matrix assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS). A shift to lower molecular masses of approximately 1500 Da for the enzymatically treated protein, compared with the intact glycoprotein, was found. The separation method was validated for linearity and reproducibility of migration time and peak area and optimized in terms of buffer pH, capillary temperature and borate concentration. This technique is sensitive to analyze batch‐to‐batch consistency in production and to test the stability of galenical formulations. After antibody storage in glass vials for 3 months at 37°C, the separation profile changed distinctly due to degradation at the carbohydrate or sialic acid moiety of the antibody, as indicated by MALDI‐TOF‐MS.
Detection and quantitation of specific mRNAs by ribonuclease protection assay using denaturing horizontal polyacrylamide gel electrophoresis: A radioactive and nonradioactive approach
Authors: Plath Annette , Peters Friedolf , Einspanier Ralf
Published: May 2005

A radioactive (32P) and nonradioactive (digoxigenin) ribonuclease protection assay (RPA) has been developed to detect mRNAs of housekeeping proteins and growth factors. A modification of polyacrylamide gel electrophoresis (PAGE) to simplify RPA is described. Both Cleangels (Pharmacia) and laboratory‐cast polyacrylamide gels, in a denaturing, horizontal electrophoresis system, were used. The amount of toxic chemicals and waste was reduced, in comparison with sequencing gels normally used for RPA. The protected RNA fragments were shown to be well‐separated, with sufficient sensitivity in this modified, quick gel system.
Isoelectric focusing and titration curves in biomedicine and in agrofood industries: A multimedia teaching program
Authors: Cade‐Treyer Denise , Cade Ambroise , Darjo Alain , Jouvion‐Moreno Myrian
Published: May 2005

The aim of this 45 min, 60 megabyte, modular program is to initiate students, scientists and engineers of biotechnology, biomedicine and agrofood industries into isoelectric focusing (IEF) and titration curves for analytical (e.g. IEF, zone electrophoresis, isotachophoresis, electrotransfer) and preparative (e.g. ionexchange chromatography, chromatofocusing) application of charge‐dependent methods. For advanced teaching, the following theoretical and practical aspects may be of interest: pH gradient engineering, IEF resolving power, generation of pH gradient, sample‐ampholyte interactions, pH gradient drift, immobilized pH gradients (IPG), IPG‐two‐dimensional (2‐D) electrophoresis, preparative methods with multi‐compartments and IPG membranes, capillary IEF, isozyme analysis, etc.). The program associates fixed and animated drawings, and computer‐assisted simulations, with spoken and written commentaries (in English). It is illustrated with numerous IEF gel patterns and titration curves and some video sequences to be run on a multimedia PC with MS Windows 3.1 (or later releases) as the only software. The linear presentation of the program may be used directly on the PC, or may be projected on a screen from the PC, for small classes or for a larger audience (200 persons). Its development as an interactive multimedia program is in progress and will soon be available on the Internet.
Determination of the number of cysteine residues in high molecular weight subunits of wheat glutenin
Authors: Morel Marie‐Hélène , Bonicel Joëlle
Published: May 2005

A simple method allowing the determination of the number of cysteine residues of the high molecular weight glutenin subunits (HMW‐GS) is presented. The method was adapted from that of Creighton (T. E. Creighton, Nature 1980, 2840, 487–489) with modified reagents for alkylation of the cysteine residues and the electrophoretic system. The acid‐urea‐polyacrylamide gel electrophoresis (PAGE) method, developed by Morel (M. H. Morel, Cereal Chem. 1994, 713, 238–242), was adopted and mixtures of iodoacetic acid and 4‐vinylpyridine were used to alkylate the glutenin subunits. The accuracy of the method was checked with some HMW‐GS whose number of cysteine residues was already known from molecular biology approaches. In subunits 5*, 2.2, 2.2* and 4.1 (of Dx types) from cultivars Ben and Fiorello, MG 7249, MG 315, and Kador, respectively, only 4 cysteine residues were demonstrated. Subunit 20 was found to exist as a regular Bx and By subunit pair, with the Bx subunit containing only 2 cysteine residues, which is unusual.
Characterization of natural peptides from bovine tissue using capillary electrophoresis, high performance liquid chromatography, matrix‐assisted laser desorption ionization, and Edman degradation
Authors: Weiler Angelika , Stoeva Stanka , Lehmann Rainer , Grübler Gerald , Paulus Gerd , Voelter Wolfgang
Published: May 2005

A combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) spiking, Edman degradation, amino acid analysis and matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) was applied for the analysis of the primary structure of β‐thymosins. CE has been used to resolve peaks which coelute on HPLC, as well as to help establish the identity of tryptic fragments in the peptide mapping experiments of the two highly homologous β‐thymosins.