bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K‐12
Authors: Link Andrew J. , Robison Keith , Church George M.
Published: May 2005

Mining the emerging abundance of microbial genome sequences for hypotheses is an exciting prospect of “functional genomics”. At the forefront of this effort, we compared the predictions of the complete Escherichia coli genomic sequence with the observed gene products by assessing 381 proteins for their mature N‐termini, in vivo abundances, isoelectric points, molecular masses, and cellular locations. Two‐dimensional gel electrophoresis (2‐DE) and Edman sequencing were combined to sequence Coomassie‐stained 2‐DE spots representing the abundant proteins of wild‐type E. coli K‐12 strains. Greater than 90% of the abundant proteins in the E. coli proteome lie in a small isoelectric point and molecular mass window of 4–7 and 10–100 kDa, respectively. We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone. Of the 223 uniquely identified loci, 60% of the encoded proteins are proteolytically processed. As previously reported, the initiator methionine was efficiently cleaved when the penultimate amino acid was serine or alanine. In contrast, when the penultimate amino acid was threonine, glycine, or proline, cleavage was variable, and valine did not signal cleavage. Although signal peptide cleavage sites tended to follow predicted rules, the length of the putative signal sequence was occassionally greater than the consensus. For proteins predicted to be in the cytoplasm or inner membrane, the N‐terminal amino acids were highly constrained compared to proteins localized to the periplasm or outer membrane. Although cytoplasmic proteins follow the N‐end rule for protein stability, proteins in the periplasm or outer membrane do not follow this rule; several have N‐terminal amino acids predicted to destabilize the proteins. Surprisingly, 18% of the identified 2‐DE spots represent isoforms in which protein products of the same gene have different observed pI and Mr, suggesting they are post‐translationally processed. Although most of the predicted and observed values for isoelectric point and molecular mass show reasonable concordance, for several proteins the observed values significantly deviate from the expected values. Such discrepancies may represent either highly processed proteins or misinterpretations of the genomic sequence. Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers, and that IcdA exists as at least three isoforms even under conditions in which covalent modification is not predicted. We enriched for proteins based on subcellular location and found several proteins in unexpected subcellular locations.
‘Proteomic contigs’ of Mycobacterium tuberculosis and Mycobacterium bovis (BCG) using novel immobilised pH gradients
Authors: Urquhart Brooke L. , Atsalos Thelma E. , Roach Daniel , Basseal David J. , Bjellqvist Bengt , Britton Warwick L. , Humphery‐Smith Ian
Published: May 2005

Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette‐Guérin (BCG) vaccine in recent trials has prompted a search for potential replacements. Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives. Novel acidic (pH 2.3–5) and basic (pH 6–11) IPG gel gradients were employed in conjunction with commercially available pH 4–7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two‐dimensional (2‐D) gels of Mycobacterium species. A total of 772 and 638 protein spots were observed for M. bovis BCG and M. tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4–7 and 6–11. Of interest was the bimodal distribution observed for proteins separated from M. bovis BCG across both Mr and pH ranges. Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes. Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis. The latter is of major importance to epidemiological studies and for patient management.
The proteome of Salmonella enterica serovar typhimurium: Current progress on its determination and some applications
Authors: O'Connor C. David , Farris Michele , Fowler Richard , Qi Shu‐Yun
Published: May 2005

Salmonella typhimurium (official designation Salmonella enterica serovar Typhimurium) is an enteric pathogen and a principal cause of gastroenteritis in humans. A comprehensive description of the proteins of Salmonella and their patterns of expression under different environmental conditions would greatly increase our understanding of the virulence of this organism at the molecular level and provide insights into many other aspects of Salmonella biology. While a variety of two‐dimensional studies of Salmonella have been previously carried out to address specific questions, little systematic information is available at the protein level on the numbers of Salmonella polypeptides that have homologues in other organisms, their abundance, and the frequency of post‐translational modifications. To test the feasibility of determining the proteome of Salmonella, the identities of 53 randomly sequenced cell envelope proteins have been determined by N‐terminal sequencing of spots from two‐dimensional gels. In addition to confirming the existence of previously hypothetical proteins predicted from genomic sequencing projects, we found that approximately 20% of the proteins had no matches in sequence databases. The results suggest that proteome anlysis is an efficient way to identify novel proteins from prokaryotes and that the analysis provides a useful approach to the study of Salmonella virulence.
DNA profiling resolves the limited importance of flower colour in defining varieties of Lantana camara
Authors: Scott Leon Jonathan , Graham Glenn Charles , Hannan‐Jones Martin , Yeates David Keith
Published: May 2005

Randomly amplified polymorphic DNA (RAPD), successfully used to establish flower colour, is of limited importance in characterising weedy varieties of Lantana camara. Initially the internal transcribed spacer 1 (ITS1) region was sequenced for common pink and common pink‐edged red varieties of L. camara from Australia and L. urticofolia from the neotropics. This proved unhelpful in differentiating varieties due to a lack of variation and the hybrid origin of L. camara, necessitating the utilisation of DNA profiling techniques. Unweighted pair group method arithmethic average (UPGMA) analysis of RAPD data demonstrated that geographical proximity contributes more significantly to genetic relatedness than flower colour. Analysis of molecular variance (AMOVA) likewise demonstrated that geography accounts for a relatively large variance component. These data indicate that the use of flower colour as a primary identification tool needs to be reevaluated. The use of RAPD may prove useful in characterising the weedy varieties of lantana present in Australia and the South Pacific. Since biological control efforts are being hindered by the inadequacy of current morphological taxonomy, it is expected that DNA profiling will underpin continuing studies on the management and control of L. camara.
Genetic diversity at six short tandem repeat loci within the state of Victoria, Australia
Authors: Wilson‐Wilde Linzi M. , van Oorschot Roland A. H. , Mitchell Robert J.
Published: May 2005

A new multiplex PCR system, developed by the Forensic Science Service (FSS) in the United Kingdom, permits the coamplification and typing of six short tandem repeat (STR) loci: HUMFGA, D8S1179, HUMTHO1, HUMvWA, D18S51, D21S11 and the sex determining marker Amelogenin. Data are presented on these six STRs for two populations in the state of Victoria, Australia: Caucasian and Asian. Whilst several worldwide databases are already available for the STR loci HUMTHO1 and HUMvWA, only relatively few databases exist for D8S1179, D18S51, D21S11 and HUMFGA. Allele frequencies at each locus displayed some fluctuations between the two populations. This is particularly so for HUMTHO1. Generally, however, the most common allele at each locus was the same in all populations, at all loci. A novel D8S1179 allele was found in Asians, provisionally identified as allele 19 Results for the six loci were compared with similar data from three UK resident populations: Caucasian, Afro‐Caribbean and Asian (Indian/Pakistani) populations. These indicated that ethnically similar populations display similar allele frequencies, while the Australian Asian and UK Afro‐Caribbean were found to be distinct.
A paternity case with apparently conflicting multilocus and single‐locus DNA typing results
Authors: Kühnau Wolfgang , Böhm Ingolf , Krawczak Michael , Schmidtke Jörg
Published: May 2005

A paternity case is presented in which DNA typing with thirteen variable number of tandem repeat (VNTR) systems was in apparent conflict with an initial multilocus DNA analysis using oligonucleotide probe (CAC)5. A dual approach was necessary, because the multilocus analysis yielded a proportion of aberrant nonmaternal offspring bands far too small and level of band‐sharing too high for non‐paternity. The case could finally be solved by reference to the joint likelihood ratio combining both approaches. Arguments are presented that summation of log likelihoods is indeed valid even if no information is available regarding linkage between the VNTR loci and the loci contributing to a multilocus DNA fingerprint.
High microvariation sequence polymorphism at short tandem repeat loci: Human beta‐actin related pseudogene as an example
Authors: Miścicka‐Śliwka Danuta , Grzybowski Tomasz
Published: May 2005

The human beta‐actin related pseudogene (HUMACTBP2) seems to be one of the most informative microsatellite markers known because of the high number of length and sequence variants. A total of 50 alleles found in white Caucasians from the Pomerania‐Kujawy region of Poland were analyzed by automated sequencing. In addition to STR length polymorphism, seven different types of sequence variation were observed. Alleles ranging in size between 233 and 273 bp showed regular sequence structure with tetranucleotide repeats AAAG. In the alleles ranging in size from 275 to 323 bp, hexamer units AAAAAG or AGAAAG occurred in the repeat region in addition to AAAG repeats. Two alleles (317 and 321 bp) contained two hexamers in the repeat region. There was considerable polymorphism of the hexamer position leading to allelic variants of the same size but different sequence structures. A large amount of variation in both 5′ and 3′ flanking regions was also observed. Allelic designation based on the number of all types of units within the repeat region (including the hexamer unit) is proposed. An allelic ladder composed of 21 sequenced alleles was constructed to add precision and accuracy to the identification of alleles at ACTBP2 locus.
Variation at three short tandem repeat (STR) loci in Australians: Forensic and ethnic considerations
Authors: Kaska Danuta E. , van Oorschot Roland A. H. , Mitchell Robert J.
Published: May 2005

We report the results of typing three short tandem repeat (STR) polymorphisms by a multiplex system in three forensically relevant populations of Australia; (i) a general Australian, (ii) a Caucasian, and (iii) an Asian sample. The STRs are tetranucleotide repeats in the following autosomal genes; CSF1PO, TPOX and THO1, and collectively comprise the CTT multiplex (Promega). In all three population samples, each of the loci exhibited genotype frequencies consistent with those expected under Hardy‐Weinberg equilibrium, and, further, there was no significant deviation from random association of alleles across separate loci. These two characteristics, plus the high discrimination power values, demonstrate that the CTT multiplex system is a powerful tool for DNA profiling in Australian jurisdictions. Australian Asians and Caucasians exhibit frequencies of alleles at these three STR loci very similar to those in other Asian and Caucasian samples. While most of the variation exists within any single population, there are still considerable differences between Asians and Caucasians. This difference between the two groups is minimal with respect to TPOX and greatest for THO1 alleles.
DNA analysis in disputed parentage: The occurrence of two apparently false exclusions of paternity, both at short tandem repeat (STR) loci, in the one child
Authors: Gunn Peter R. , Trueman Katherine , Stapleton Patricia , Klarkowski Derryck B.
Published: May 2005

In cases of disputed paternity investigated by DNA analysis, it is generally accepted that at least two independent exclusions be observed before concluding that a putative father is not the biological father of the child. We report here a case in which two apparent exclusions of paternity were obtained (at the loci FESFPS and TPOX), from a total of nineteen loci examined (short tandem repeats, AMFLPs, DQA1, Polymarkers). The combined paternity index of the other seventeen loci, and of two multilocus probes (33.15 and 33.6) would exceed twenty‐eight trillion to one. In the absence of any alternative putative father (including close relatives of the man tested), we concluded that the most likely explanation for these results was that the putative father was indeed the biological father, but that two independent mutations had occurred in either the ovum and/or spermatozoon. We have altered our internal laboratory criterion for exclusion of paternity to require exclusions in three independent loci.
Repetitive DNA sequences as an insight into Aeglidae (Crustacea, Anomura) evolution
Authors: D'Amato María Eugenia , Corach Daniel
Published: May 2005

The evolutionary relationships of five Atlantic Aeglidae species (Aegla neuquensis affinis, A. humahuaca, A. jujuyana, A. platensis, and A. uruguayana) were studied by (i) satellite DNA analysis using a restriction enzyme digestion and hybridization pattern approach and (ii) genome screening by using randomly amplified polymorphic DNA (RAPD) typing. The identical restriction patterns and intense interspecific hybridization patterns obtained in this study strongly suggest a recent cladogenetic event for the Aeglidae. The species‐specific amplification products which were detected using RAPD markers allowed species characterization. A total of 49 amplification products were used to construct trees by cluster analysis. The new scheme agrees in part with previous proposals based on biogeography and morphology. We considered that the subdivision northwestern‐platensis species was probably due to the rising of the Andes, which started in the Middle Miocene. Divergence due to altitude is suggested by the different altitudinal distribution of three northwestern species along the same river. The possible role of selection by ecological factor/s was observed at the population level in A. jujuyana, which has a wider altitudinal range distribution. RAPD markers revealed a high level of intraspecific diversity and important genetic flow among populations. However, a few markers showed significant differences in frequency or H between the lowermost population and the other populations, located in a different biogeographical region. The differences were not in relation to geographical distance, and we interpreted them as being due to selection. Repetitive sequences constitute an important reservoir of genetic variation, and these results show their usefulness in testing and proposing evolutionary hypothesis in crabs. These sequences seem to have played an important role in aeglid evolution. Ecological factors related to altitude have probably influenced macro‐ and microevolutionary processes, at least in northwestern species.
Assessment of genetic diversity by DNA profiling and its significance in silkworm, Bombyx mori
Authors: Nagaraju Javare Gowda , Singh Lalji
Published: May 2005

Silkworm genetic resources that are being maintained in different countries are yet to be adequately tapped to develop elite varieties that are suited to different agro‐eco‐climatic conditions of countries like India. This is mostly due to unavailability of efficient protocols that could uncover usable genetic variability in silkworms. Molecular markers are known to provide unambiguous estimates of genetic variability of populations since they are independent of confounding effects of environment. The DNA fingerprinting assays, based on random amplified polymorphic DNA (RAPD) and banded krait minor satellite DNA (Bkm) 2(8) multilocus probes, which successfully characterise the diverse silkworm genotypes at their DNA level, are described. The use of these two DNA fingerprinting assays in estimation of within‐ and between‐population genetic diversity is discussed.
Informative enzyme/probe combinations for the multilocus DNA fingerprinting of marsupials
Authors: Lambert Kim , Jones Michelle , Shimmin Glenn , Sofronidis George , Bowden Don , Taggart David , Temple‐Smith Peter
Published: May 2005

DNA fingerprinting has become an invaluable tool in the study of population genetics, paternity success, and individual identification; however, the species specificity of some methods has made the wide‐range screening of many different species very time‐consuming. In this study we describe the development and application of reliable and informative DNA fingerprinting techniques in a range of marsupial species using three different restriction enzyme and two oligonucleotide probe combinations. Six species from four marsupial families, the koala (Phascolarctidae), tammar wallaby (Macropodidae), southern hairynosed wombat (Vombatidae), kowari, and dusky and brown marsupial mice (Dasyuridae) were examined. Restriction enzymes HinfI AluI and HaeIII were used in combination with the digoxygenin (DIG)‐labelled oligonucleotide probes (CAC)5 and (GGAT)4. The combinations of HinfI/(GGAT)4, AluI/(CAC)5 and AluI/(GGAT)4 were the most informative, providing highly resolved bands, low background, and the lowest band sharing between individuals. The genetic diversity evident within the different species showed a clear relationship between the level of band sharing and population size. The greatest levels of band sharing were found in the kowaris (80%), which were part of a long‐term captive colony originating from a few founders, and the lowest levels of band sharing were found in the marsupial mice (30–35%) and tammar wallaby (45%), which were caught from large outbred wild populations.
Microsatellite‐based cancer detection using capillary array electrophoresis and energy‐transfer fluorescent primers
Authors: Wang Yiwen , Hung Su‐Chun , Linn Jürgen F. , Steiner Gabriel , Glazer Alexander N. , Sidransky David , Mathies Richard A.
Published: May 2005

The development of sensitive, rapid, and accurate methods and apparatus for high‐throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR‐based bladder cancer diagnosis can be performed using capillary array electrophoresis and two‐color labeling with energy‐transfer (ET) fluorescent primers. Rapid (≤35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of ± 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.
Capillary electrophoresis for diagnosis of metabolic disease
Authors: Elgstoen Katja B. Presto , Jellum Egil
Published: May 2005

Capillary electrophoresis (CE) equipped with a diode‐array detector have been used to determine diagnostic metabolites occurring in urine of patients with various diseases. The urine samples were injected directly onto the CE instrument without any pretreatment. Identification of abnormal metabolites was based on relative migration times and characteristic diode‐array spectra. The CE‐method readily diagnosed alkaptonuria, neuroblastoma and liver failure due to galactosemia. The simple and automated CE method appears to be suitable for implementation as a screening test in analytical systems aimed at diagnosis of metabolic disorders.
Enhancement of resolution of low molecular weight RNA profiles by staircase electrophoresis
Authors: Cruz‐Sánchez José M. , Velázquez Encarna , Mateos Pedro F. , Velázquez Enrique , Martínez‐Molina Eustoquio
Published: May 2005

Stable low molecular weight (LMW) RNA comprises molecules used in the taxonomy of microorganisms and in studies on the microbial diversity of populations. However, the use of electrophoretic techniques has been hampered due to the low resolution obtained with techniques used for the separation of this kind of molecule. In this work we develop an electrophoretic method (staircase electrophoresis) that increases the resolution of the technique. This improvement in the resolution adequately resolves the three zones that integrate the profiles of LMW RNA: ribosomic 5S RNA (5S rRNA), class 2 transfer RNA (tRNA), and class 1 transfer RNA, allowing the technique to be applied to taxonomic studies (diagnostic, the identification of the individuals), phylogenetic studies and studies on naturally occurring microbial populations.
Reorientation of large DNA molecules in concentrated polyacrylamide solution during crossed‐field electrophoresis
Authors: Oana Hidehiro , Doi Masao , Ueda Masanori , Yoshikawa Kenichi
Published: May 2005

Recently, we found that, in concentrated neutral solutions, DNA molecules migrate in linear conformation under steady electric field. In this paper, we report the conformational change of DNA during 120° crossed‐field electrophoresis in the same polymer solution. We found that, in concentrated polyacrylamide solutions, the reorientation process of DNAs becomes simple: the DNA goes back along the previous track and the reorientation time is longer motion for the separation of DNA fragments in pulsed field gel electrophoresis. We expect that this phenomenon is useful for a more efficient separation technique of large DNAs than the current pulsed field gel electrophoresis.
D12S67, a bipartite locus: Differential amplification of parts of the nucleotide sequence reveals considerable polymorphism
Authors: Minaguchi Kiyoshi
Published: May 2005

The STR system D12S67 was amplified by polymerase chain reaction (PCR) and analyzed by denaturing gel electrophoresis followed by silver staining. Among 133 DNA samples from Japanese individuals, 11 alleles were observed and the heterozygosity was 80%. When sequences of the alleles were compared, each allelic segment contained 35–45 gata or gaca tetranucleotide repeats. Although a (gaca)n repeat block was concentrated in a defined region, nine different blocks of (gata)n repeats were observed separated by the (gaca)n repeat and single copy sequences. In addition, the allelic differences result from the total number of repeats of at least three (gata)n and the (gaca)n repeat regions. Novel primers overlapping part of the sequence with each other were constructed in the center of the D12S67 sequence, and the 5′ and 3′ segments were amplified by PCR. Both of these segments were highly polymorphic and showed heterozygosities of 77 and 78% with 7 and 10 alleles, respectively. The genotypes of the two polymorphisms were not concordant with the original polymorphism and the combination of the 5′ and 3′ side segment polymorphisms enabled further detailed classification of the D12S67 locus by simple comparison of the PCR product sizes. The number of the allele types increased up to 35 and the heterozygosity to 93%.
Ampholyte dissociation theory and properties of ampholyte aqueous solutions
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

Two different approaches (stepwise and parallel mechanism) for describing the dissociation of amphoteric protolytes are described. Additionally, the classification of the possible model on the basis of the ampholyte lifetime state is proposed. The possibility of application of different schemes is discussed. It is suggested that the correct description may be based only on highly relaxing models. The formulas describing the properties of ampholyte solutions are derived for the case of two ionogenic groups. It is demonstrated that the incorrect choice of the dissociation model may result in essential mistakes in calculation of such values as buffer capacity, electrophoretic mobility, and conductivity. On the basis of the theory here developed, the interpretation of natural pH‐gradient conductivity is given. Some terminology questions are also discussed.
Gel electrophoresis of DNA fragments in narrow‐bore capillaries
Authors: Lindberg Peter , Stjernström Mårten , Roeraade Johan
Published: May 2005

In this work, we studied the behavior of electrophoretic columns, having an inner diameter (ID) of 2–10 μm, filled with a cross‐linked polyacrylamide gel matrix. The usefulness of these columns for DNA sequencing is discussed. Evaluation of column performance included tests of gel stability and migration time reproducibility. Confocal laser‐induced fluorescence (LIF)‐detection was employed utilizing a 488 nm argon ion laser for separations of C‐ and T‐terminated DNA Sanger fragments. Reducing the inner diameter of the column from 50 μm to 10 μm resulted in an approximately eightfold increase in lifetime, under conditions in which the columns were subjected to a field strength of 1000 V/cm. The 10 μm ID columns were utilized for separation of Sanger fragments, and adequate detection sensitivity was obtained by stacking of the fragments from a deionized sample solution. A linear algorithm for retention data synchronization between individual electropherograms was employed to provide a route towards a reliable automated base calling protocol.
Capillary electrophoresis of DNA restriction fragments: Effect of polymer properties
Authors: Braun Björn , Blanch Harvey W. , Prausnitz John M.
Published: May 2005

The mechanism of DNA separation by dilute polymer solutions in capillary electrophoresis is not well understood. To provide information on the effect of polymer properties on DNA separations, four polymers that differ in size, shape and stiffness were examined. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp – 23 130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp; star‐(polyethylene oxide) and linear poly(ethylene oxide) provide separation of fragments to 1353 bp.
Sodium dodecyl sulfate‐polymer capillary electrophoresis for the analysis of cell culture proteins
Authors: Klyushnichenko Vadim , Kula Maria‐Regina
Published: May 2005

The application of sodium dodecyl sulfate (SDS)‐polymer capillary electrophoresis (CE) to the analysis of proteins of hybridoma cell culture supernatant is demonstrated. All steps of the developed analysis are shown and discussed. Following optimization of sample preparation and concentration, as well as of the mode of injection all main protein components were separated with high resolution and selectivity. Proteolytic degradation of IgG, due to microbial contamination, was observed on SDS‐gel CE and capillary zone electrophoresis (CZE). The technique was applied to monitor the cell cultivation process and concentrations of main components are demonstrated. IgG was found to increase from initially 5 mg/L to 20 mg/L after 4 days and to > 130 mg/L after 14 days of cultivation. The techniques may be applied to other biotechnological processes for the production or purification of proteins.
Capillary electrophoretic separation of structurally similar solutes in noncross‐linked poly(acrylamide‐co‐N‐isopropylacrylamide) solution
Authors: Sawada Hirokazu , Jinno Kiyokatsu
Published: May 2005

Noncross‐linked acrylamide (AA)‐N‐isopropylacrylamide (IPAAm) copolymers have been used as a buffer additive in capillary electrophoretic separation of structurally similar small solutes. Seven kinds of barbiturates and five kinds of dansylated (Dns) amino acids, which have different hydrophobic side chains, were separated on poly(AA‐co‐IPAAm)‐filled columns and on AA polymerfilled columns under the condition of totally eliminating the contribution of electroosmotic flow (EOF). It is known that the copolymer containing IPAAm has thermosensitive properties, and the hydrophobicity of its surface changes with surrounding temperature. In this investigation, therefore, the effects of an isopropyl group in the copolymer on the electrophoretic separation of the small solutes were studied by comparing the two polymer‐filled columns at ambient temperature and at elevated temperature. Although slight differences in migration behavior were observed at ambient temperature between the columns filled with these two polymer solutions, obvious differences in the separation of the solutes were observed at elevated temperature. The observed changes on the migration behavior might be caused by the interaction between copolymer chains exhibiting hydrophobic property and the solute.
Capillary isoelectric focusing
Authors: Rodriguez‐Diaz Roberto , Wehr Tim , Zhu Mingde
Published: May 2005

Capillary isoelectric focusing (CIEF) provides excellent resolution of proteins with the advantage that separations are carried out in a capillary format with on‐tube detection and automated analysis. Recent advances in the technique provide improved resolution, reproducibility and reliability. This review summarizes improvements in sample preparation, capillary selection, and focusing and zone mobilization procedures which have helped CIEF become a more robust analytical method. New applications are reviewed, in particular the use of CIEF in analysis of biopharmaceutical products.
Chiral capillary electrophoresis with noncyclic oligo‐and polysaccharide chiral selectors
Authors: Sutton Richard M. C. , Sutton Karen L. , Stalcup Apryll M.
Published: May 2005

A number of noncyclic oligo‐ and polysaccharides have been used as chiral additives in capillary electrophoresis (CE). This review offers a broad survey of the types of oligo‐ and polysaccharides which have been investigated and also some of the conditions developed for the enantioseparation of a wide range of drugs and other racemic compounds. Details of enantioseparation mechanisms are also discussed, in addition to some of the parameters required for optimization of the enantioresolution of chiral molecules.
Capillary electrophoresis of pesticides
Authors: El Rassi Ziad
Published: May 2005

Pesticides are important and diverse environmental and agricultural species. Their determination in pesticide formulations, in feed and food, and in complex environmental matrices (e.g., water, soil, sludge, sediments, etc.) often requires separation methods of high efficiency, unique selectivity and high sensitivity. As shown in this comprehensive review, capillary electrophoresis meets these requirements and has proved to be a suitable microseparation technique for the analysis of a wide variety of chiral and achiral pesticides. It is also shown that by combining selective precolumn derivatization schemes, sensitive detection methods (e.g., laser induced fluorescence detection) and trace enrichment techniques, capillary electrophoresis (CE) is capable of determining pesticides at trace levels as those usually encountered in environmental samples.
High‐resolution density gradient electrophoresis of proteins and subcellular organelles
Authors: Tulp Abraham , Verwoerd Desirée , Benham Adam , Neefjes Jacques
Published: May 2005

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011–1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, Ø 2.2 cm). At pH 8.66 and 250 V, β‐lactoglobulin (Mr 36 600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76 000–81 000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30 000) separated into its isoenzymes. From these results we arrive at the term high‐performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well‐separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse‐chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.
Rab proteins and post‐Golgi trafficking of rhodopsin in photoreceptor cells
Authors: Deretic Dusanka
Published: May 2005

Polarized sorting of rhodopsin in retinal rod photoreceptors is mediated by post‐Golgi vesicles that bud from the trans‐Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. This domain surrounds the cilium that connects the inner segment and the rod outer segment to which mature rhodopsin is delivered. To dissect the sorting machinery that regulates budding, targeting, and fusion of rhodopsin carrier vesicles, their GTP‐binding protein composition has been studied using multiple means including high‐resolution two‐dimensional gel electrophoresis and [32P]GTP overlays of renatured proteins. These studies indicate a succession on rhodopsin‐bearing vesicles of rab6, rab11, rab3 and rab8, all members of the small GTP‐binding protein family of the known regulators of membrane trafficking. In this review the role of rab proteins in post‐Golgi trafficking of rhodopsin is discussed.
Preparative two‐dimensional gel electrophoresis of membrane proteins
Authors: Pasquali Christian , Fialka Irene , Huber Lukas A.
Published: May 2005

Electrophoretic techniques, and especially two‐dimensional gel electrophoresis (2‐DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well as for the identification of protein‐protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first or second dimension of 2‐DE. Here we describe the modification of a protocol using a combination of 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4‐dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver‐stained gels obtained from identical samples (4 μg). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale‐up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialkaet al., Electrophoresis1997,18, 2582–2590).
Insulin‐regulatable phosphoproteins in 3T3‐L1 adipocytes form detergent‐insoluble complexes not associated with caveolin
Authors: Hill Michelle M. , Clark Sharon F. , James David E.
Published: May 2005

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin‐regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, some of these intermediates, including IRS‐1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high‐speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3‐L1 adipocytes. A large proportion of adipocyte phosphoproteins co‐fractionated in the HSP fraction. In an attempt to resolve insulin‐regulatable phosphoproteins, we subjected 32P‐labeled subcellular fractions to two‐dimensional gel electrophoresis (2‐DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X‐100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin‐regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent‐insoluble signaling complexes.
Representation of amino acid sequences as two‐dimensional point patterns
Authors: Pleißner Klaus‐Peter , Wernisch Lorenz , Oswald Helmut , Fleck Eckart
Published: May 2005

An algorithm for the representation of amino acid sequences as two‐dimensional point patterns (2‐D plot) is described. The algorithm is based on chaos game representation (CGR) for DNA sequences and was extended for amino acid sequences. The 2‐D plot depicts the sequentiality of amino acids and the amino acid composition of a protein. Changes in a protein sequence as insertion, deletion and repeats of amino acids are characterized by specific geometrical properties and changes in the 2‐D plots. The 2‐D plot may be considered as a two‐dimensional “fingerprint” of a protein. The properties of the algorithm are explained by user‐defined amino acid sequences. As an example the 2‐D plots of two selected heart proteins are generated. The sequences of these proteins are obtained from the protein sequence database SWISS‐PROT.
Restriction sites as identification tags for lymphocyte cDNAs
Authors: Frey Johann R. , Fol Bruno , Lefkovits Ivan
Published: May 2005

A cDNA library was prepared from BW 5147 murine lymphoma cells in λ ecc III phage and randomly partitioned into 291 sectors, each with 800–1000 recombinant phage plaques. One sector was chosen for further characterization in terms of sensitivity to restriction endonuclease cutting. Aliquots of DNA preparations from this sector were treated with XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI before being used as templates in a cell‐free expression system. The polypeptide products were separated by two‐dimensional (2‐D) gel electrophoresis and radiofluorographs of the gels were submitted to computer‐aided image analysis. The matched patterns were inspected for the presence or absence of spots upon individual endonuclease treatments. Thereafter the results were integrated in a data matrix which served as a basis to construct “restriction tags” for all spots. These (restriction) tags are binary numbers termed “cut numbers” and are a representation of the set of recognition sequences which are (or are not) part of the coding sequence. From 493 sequences (visualized as 2‐D gel spots), 12 were not cut by any of the nine enzymes, while 45 were cut by all of them. The percentages of sequences resistant to enzyme treatment ranged between 17% and 77% for NcoI and XhoI, respectively. The enzyme treatments led to the appearance of a certain portion of “new spots”, probably products from truncated sequences. From 512 possible cut numbers, 136 were assigned to the 493 spots. Restriction tags are available to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.
Protein profiling in cardiac tissue in response to the chronic effects of alcohol
Authors: Patel Vinood B. , Corbett Joseph M. , Dunn Michael J. , Winrow Vivienne R. , Portmann Bernard , Richardson Peter J. , Preedy Victor R.
Published: May 2005

An investigation was made into cardiac protein levels after chronic ethanol consumption to examine whether specific proteins are affected by alcohol. Ethanol was administered for six weeks to male Wistar rats which were fed a nutritionally complete liquid diet containing 35% of total calories as ethanol. Controls were pair‐fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose; thus both groups had identical nutritional intakes, albeit differences in ethanol or carbohydrate. After six weeks' feeding, cardiac tissue was removed and analyzed by two‐dimensional electrophoresis, where equal amounts of proteins were studied. Protein patterns were analyzed by computerized densitometry and characterized by comparison with a database of known cardiac proteins. Chronic alcohol feeding caused significant decreases in the relative amounts of various proteins including several tentatively identified as heat shock protein (HSP) 60, HSP70, and desmin. The relative proportions of actin, vimentin, myosin light chain 1, myosin light chain 2, and albumin, remained unchanged. Examination of antibodies raised against HSP65 showed no overt differences in plasma levels following chronic alcohol consumption, and liver changes as assessed by histology were mild. In conclusion, chronic alcohol appears to have selective effects on particular proteins, and the effects were not directly ascribed to overt liver dysfunction or malnutrition. This may explain some of the functional and morphological characteristics observed in alcohol‐induced heart muscle disease, including reduced contractility.
Horizontal ultrathin‐layer multi‐zonal electrophoresis of DNA: An efficient tool for large‐scale polymerase chain reaction (PCR) fragment analysis
Authors: van den Berg Bartel M.
Published: May 2005

A rapid and efficient procedure for large‐scale analysis of polymerase chain reaction (PCR) fragments in the range of 200–3000 base pairs is presented. The procedure is based on horizontal ultrathin‐layer multi‐zonal (HUME) electrophoresis of PCR fragments in polyacrylamide gels followed by silver staining. HUME gels can be prepared rapidly using a simple procedure called the flap technique. The electrophoretic set‐up allows the use of multi‐channel pipettes for sample loading. Separation and detection of the PCR fragments from sample preparation to silver staining can be carried out in 2 h. Using four electrophoresis units, one technician can analyze 400 PCR fragment samples in 2 h.
Applicability of nonsyngeneic cell models for screening of genes in monogenetics diseases via differential display technique
Authors: Planitzer Simone A. , Machl Andreas W. , Kubbies Manfred
Published: May 2005

Conventional subtraction library techniques or DNA‐transfection studies are standard techniques applied for identification and isolation of genes relevant in monogenetic diseases like Fanconi anemia (FA). The differential display technique (DDT) was developed to compare mRNA expression between a mutant cell line and its syngeneic control and allows comparison of almost all mRNA species within a short time. However, for identification of genes relevant in monogenetic diseases, no syngeneic cell model is available. In this report, we show that the use of nonsyngeneic diploid human fibroblasts does not increase the number of differentially displayed bands due to diversity of untranslated regions. cDNA bands with a length of up to 1000 bp were obtained and applied to DDT. After screening of about 13000 cDNA bands, only 0.5% were found to be differentially expressed between FA and control cells. Finally, three mRNAs were cloned and verified in Northern blot experiments to be differentially expressed in FA fibroblasts. The low number of differentially displayed cDNA bands in DDT indicates the usefulness of this statistical, molecular approach for identification of multiple genes dysregulated in gene regulation cascades potentially relevant for cell cycle disturbances.
Sex determination of foresic of forensic samples by polymerase chain reaction of the amelogenin gene and analysis by capillary electrophoresis with polymer matrix
Authors: Pouchkarev Vladimir P. , Shved Eugene F. , Novikov Peter I.
Published: May 2005

The aim of this study was to validate an application of GenePrint™ Sex Determination System based on amplification of a section of the X‐Y homologous gene amelogenin followed by capillary electrophoresis (CE) separation of polymerase chain reaction (PCR) products for gender testing of forensic DNA. It was found that subnanogram quantities of male and female DNA were correctly detected by this system. Experiments were performed to investigate the possibility of quantitating the X‐Y chromosome‐specific PCR products to disclose sex‐mixed DNA samples. It was found that observed electrophoretic profiles correctly reflected an X‐Y chromosome proporation of the DNA sample which was introduced into the PCR mix. The tested amelogenin PCR‐CE system was successfully used for gender testing of a wide range of biological evidence including sex‐mixed DNA samples from rape cases. These results demonstrate that the tested amelogenin PCR‐CE system is a useful tool for gender determination of forensic DNA.
Rapid mitochondrial DNA typing using restriction enzyme digestion of polymerase chain reaction amplications followed by capillary electrophoresis separation with laser‐induced fluorescence detection
Authors: Butler John M. , Wilson Mark R. , Reeder Dennis J.
Published: May 2005

The polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expenses of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR‐amplified mtDNA using RsaI and Mn/I and then capillary electrophoresis (CE) to separate and size the PCR‐RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T→C transition at position 16189, which gives rise to the so‐called “C‐stretch” in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.
Oligonucleotide DNA fingerprinting optimized to determine parentage in three beetle species
Authors: Benken Theodor , Müller Josef K. , Peschke Klaus
Published: May 2005

Oligonucleotide fingerprinting was performed on three beetle species from different families (Coleoptera: Staphylinidae: Aleochara curtula; Silphidae: Nicrophorus vespilloides; Tenebrionidae: Blaps lethifera) to obtain detailed information on parentage in mating systems. We report variations in the number of hybridizing fragments for the different species depending on the combinations of probes and restriction enzymes used. In addition to conventional multilocus fingerprint patterns, we established a single locus system for A. curtula (GTG5; HinfI) and described an oligolocus system in N. vespilloides (GATA4; HaeIII).