bluuebunny/crossref_metadata_2025 huggingface dataset:
Enantiomer separation of denopamine by capillary electrophoresis with charged and uncharged cyclodextrins
Authors: Ishibuchi Kiyotaka , Izumoto Shin‐ichi , Nishi Hiroyuki , Sato Tadashi
Published: May 2005

Direct separation of enantiomers of denopamine was investigated by capillary electrophoresis employing charged and uncharged cyclodextrin (CD) derivatives. Uncharged β‐type CDs, having hydrophobic groups, were essential for the enantioseparation of denopamine; of these, especially dimethyl‐β‐CD was effective. Among charged CDs, γ‐type as well as β‐type CDs were found effective for the enantioseparation of denopamine. Reversal of migration order of R‐form (active) and S‐form enantiomers was investigated by using two types of coated capillaries: (i) an amine capillary with an inner wall coated with dimethylamino groups, and (ii) a polyacrylamide‐coated capillary. Manipulation of migration order could be easily performed by selecting suitable capillaries, buffer pH, and CDs.
A set of electrophoretic molecular markers for strain typing and population genetic studies of Histoplasma capsulatum
Authors: Carter Deidre A. , Burt Austin , Taylor John W. , Koenig Gina L. , Dechairo Bryan M. , White Thomas J.
Published: May 2005

A set of eleven biallelic and three multiallelic molecular markers have been developed to analyze populations of Histoplasma capsulatum. All markers are amplified by polymerase chain reaction (PCR) and can be readily scored using minimal amounts of template DNA. The 11 biallelic loci have polymorphic restriction endonuclease sites or small insertions or deletions which may be assessed by agarose gel electrophoresis. These markers are inherited in an unambiguous manner and are ideal for assessing structure and gene flow within US populations of H. capsulatum, but are monomorphic in non‐US populations. Both length and sequence variation are present in the multiallelic loci, which can be scored by direct sequencing, polyacrylamide gel electrophoresis, or single‐strand conformation polymorphism (SSCP): As they are hyper‐variable, the multiallelic loci can be used to type isolates and to assess the level of genetic variation within populations. Preliminary results indicate that the three multiallelic markers presented are sufficient to distinguish isolates at the individual level and are polymorphic in both US and non‐US populations. This collection of molecular markers will be a useful tool in population and epidemiology studies of H. capsulatum.
A simple and fast procedure for high quality DNA isolation from gels using laundry detergent and inverted columns
Authors: Pusch Carsten
Published: May 2005

A quick method for the recovery of DNA from agarose and polyacrylamide gels with high efficiency and quality is described. Excised gel slices, containing at least 10 ng DNA, are incubated for 15 min at 60°C in the presence of laundry detergents. An “inverted column” is obtained by covering the extraction liquid by a layer of cotton wool, Sephadex G‐50 and another layer of cotton wool. Following centrifugation the supernatant containing the DNA is recovered by aspiration and the DNA is precipitated with isopropanol and tRNA, washed with ethanol and air‐dried. The yield of reisolated DNA does not depend on DNA fragment size and small quantities of gel volume. Thus, the technique may prove useful in a broad range of applications in the methodology of molecular biology.
Oriented macroporous polyacrylamide gels
Authors: Charlionet Roland , Machour‐Merlet Nadine , Leclerc Stéphane , Malandain Jean‐Jacques
Published: May 2005

Macroporous gels with huge cavities and partition walls result from controlled microsyneresis during gelation. In this report we show that the microsyneresis process can be further controlled: it is possible to orient the partition walls of macropores by the use of an electric field throughout polymerization. Our oriented macroporous polyacrylamide gels offer a specific structure: a set of parallel channels of about 2 μm inner diameter, with polyacrylamide walls including acidic groups linked to the aggregated matter.
Fast separation of underivatized carbohydrates by coelectroosmotic capillary electrophoresis
Authors: Zemann Andreas , Nguyen Duc Tuan , Bonn Günther
Published: May 2005

A method for the rapid quantitative analysis of underivatized acidic sugars, monosaccharides and disaccharides using coelectroosmotic capillary electrophoresis was developed. Indirect UV detection at 254 nm using sorbate as background electrolyte was employed for monitoring the analytes. A highly alkaline pH value of the electrolyte system was chosen in order to achieve an electrophoretic mobility of the saccharides towards the anode. A dynamic reversal of the electroosmotic flow and, by this means, a codirectional movement of the negatively charged analytes and the electroosmotic flow is accomplished by employing a polycationic surfactant (hexadimethrine bromide), which is added to the background electrolyte. To further improve the resolution of specific carbohydrates, acetone is used as organic modifier. A practical application of the developed method for the fast determination of fructose, glucose, and sucrose in various soft drinks is provided.
Separation of disaccharides as their borate complexes by capillary electrophoresis with indirect detection in visible range
Authors: Plocek Jiří , Chmelík Josef
Published: May 2005

Borate complexation was used to make possible the separation of disaccharides by capillary electrophoresis with indirect detection. A high borate concentration did not affect the indirect detection sensitivity in as negative a way as predicted previously. The concentration sensitivity for sucrose was determined to be 2 mM at the borate concentration of 200 mM in running electrolyte. The newly introduced backround chromophore, p‐nitrophenol, allows the monitoring of the separation process in a visible range at 400 nm. This also enables the indirect detection of UV‐absorbing compounds in complex mixtures in which they would be impossible to detect with a UV‐absorbing backround chromophore.
Sulfobutylether‐β‐cyclodextrin‐mediated capillary electrophoresis for separation of chlorinated and substituted phenols
Authors: Groom Carl A. , Luong John H. T.
Published: May 2005

Cyclodextrin‐mediated capillary electrophoresis has been developed for separation and analysis of chlorinated as well as substituted phenolic compounds. The procedure used a negatively charged sulfobutylether‐β‐cyclodextrin (SB‐βCD) to effect differential partitioning of the phenols between the buffer and CD phases. In 50 mM phosphate buffer containing as low as 1 mM SB‐β‐CD, 25 phenolic compounds including 11 Environmental Protection Agency (EPA) priority phenols were separated with theoretical plate numbers well above 100 000, for 50 cm of effective length in most cases. An equilibrium complexation model was used for investigating the effect of pH as well as different cyclodextrin concentrations on the electrophoretic mobility. The cyclodextrin‐mediated capillary electrophoresis system was also applicable for separating and quantifying the level of pentachlorophenol in contaminated soil samples.
Isoelectric properties of the soluble matrices in relation to the chemical composition of some Scleractinian skeletons
Authors: Dauphin Yannicke , Cuif Jean‐Pierre
Published: May 2005

Soluble matrices of four Scleractinian skeletons (Madrepora, Favia, Leptastrea and Fungia), were extracted and studied by isoelectric focusing (IEF). The main part of the extracted matrices is acidic. IEF gels are poorly stained or unstained by Coomassie Blue and silver. The positive Alcian Blue staining is indicative of a sulfated and acidic nature of the glycoproteins. Chemical analysis shows high S contents in Scleractinian skeletons. These results are supported by the comparison with IEF and chemical contents of Molluscan shells.
Proteomic ‘contigs’ of Ochrobactrum anthropi, application of extensive pH gradients
Authors: Wasinger Valerie C. , Bjellqvist Bengt , Humphery‐Smith Ian
Published: May 2005

The most extensive linear pH gradients yet employed in combination with two‐dimensional gel electrophoresis are described, along with their application in proteome analysis. A significant proportion of the protein compliment of bacterial species is believed to be accessible using an extended linear pH gradient of 2.3 to 11.0. Protein standards with predicted isoelectric points (pI) ranging from 3.24 to 9.56 were used to confirm focusing positions with respect to the immobilised pH gradients (IPG) prior to mapping studies of Ochrobactrum anthropi. Multiple gel images were used to construct contiguous windows of protein expression (‘proteomic contigs’) within 18 cm pH gradients 2.3–5, 4–7, and 6–11 in conjunction with 15%T and 7.5%T acrylamide gels, the latter being used to resolve higher molecular weight (Mr) proteins. Each IPG had a 5 cm region of similar pH gradient overlap at pH 4–5 and pH 6–7 that was used to construct an image of protein expression characteristic of whole cell lysates. This is reminiscent of genomic sequencing initiatives whereby portions are combined to form a contiguous picture of the whole. The protein maps obtained demonstrated a means of resolving the many tens of thousands of cellular proteins likely to occur in eukarvotic systems, but also highlighted the need to further optimise protein extraction, equilibration buffers, and separation conditions of higher Mr proteins occurring at extreme pI. Theoretical 2‐D protein maps were constructed for five organisms for which the total DNA sequence is now available. In all cases, higher Mr acidic and basic proteins were shown to be common.
Spontaneous and induced minisatellite instability
Authors: Jeffreys Alec J. , Bois Philippe , Buard Jérǒme , Collick Andrew , Dubrova Yuri , Hollies Caroline R. , May Celia A. , Murray John , Neil David L. , Neumann Rita , Stead John D. H. , Tamaki Keiji , Yardley Jane
Published: May 2005

Minisatellites provide not only the basis for DNA fingerprinting and DNA profiling but also extremely informative systems for analysing processes of tandem repeat turnover in the human genome. Minisatellite instability appears to involve distinct mutation processes in somatic and germline cells; in the germline, mutation is frequently dominated by inter‐allelic conversion‐like events most likely occurring at meiosis and apparently regulated by cis‐acting mutation initiator elements. Attempts to define these initiators in transgenic mice have so far been thwarted by what appears to be a major human/mouse barrier to the inter‐species transfer of repeat instability. Minisatellites not only show high frequency spontaneous mutation in the germline, but also appear to be very sensitive to mutation induction by ionizing radiation, both in experimentally irradiated mice and in human populations exposed following the Chernobyl disaster; the mechanisms of mutation induction by radiation remain enigmatic.
Mitochondrial D‐loop diversity in Australian riverine and Australian desert aborigines
Authors: van Holst Pellekaan Sheila , Frommer Marianne , Sved John , Boettcher Barry
Published: May 2005

Population structure has been revealed in mitochondrial D‐loop segment 1 (mt DLS1) sequences from Australian Aboriginal people in the Darling River region of NSW (Riverine) and from Yuendumu in central Australia (Desert). Comparison with five published global studies reveals that these Australians demonstrate greatest divergence from some Africans, least from Papua New Guinea (PNG) highlanders, and only slightly more divergence from some Pacific groups (Indonesian, Asian, Samoan, and coastal PNG). A median networks approach [1] demonstrates that several hypervariable nucleotide sites within the DLS1 are likely to have undergone mutation independently. A comprehensive evaluation of specific nucleotide variants with the large amount of global sequence data now available has been achieved in three stages of analysis: (i) identification of key nucleotide variants (from the Cambridge reference sequence [2]) in the Aboriginal Australian by pairwise comparison and construction of a ‘local’ median network, (ii) identification of key nucleotide variants in a selected global sample including Australian mtDLS1 types most different from each other, and (iii) calculation of the frequency with which these key nucleotide sites occur as variants in a greatly extended global sample. The third stage of the analysis revealed that nucleotides 16287 and 16356 are unique markers for representatives from the northern Riverine region. A ‘thymine’ at nucleotide 16223 is an informative signature of African and several identifiable non‐African DLS1 types, whereas the ‘cytosine’ form is a marker for European, Pacific, and some Asian populations.
Genome analysis of the fungal plant pathogen, Leptosphaeria maculans using pulsed field gel electrophoresis
Authors: Howlett Barbara J.
Published: May 2005

Pulsed field gel electrophoresis (PFGE), or electrophoretic karyotyping, separates chromosomal‐sized pieces of DNA in agarose gels where the orientation of the electric field is periodically altered. This technique has revealed that many fungi have a high degree of chromosomal length polymorphisms. Often the only isolates with identical karyotypes are derived from a single clone, thus PFGE provides a ‘genetic fingerprint’ for them. The size range and number of chromosomes within isolates of a particular species are usually constant, hence PFGE can distinguish between morphologically similar fungi. This technique can also be used to follow inheritance of chromosomal length polymorphisms and shows that in some fungi novel‐sized chromosomes are produced during meiosis. As well as resolving the nuclear (A‐type) chromosomes, it can also resolve dispensable (B‐type) chromosomes and cytoplasmic genomes including mitochondrial DNA and linear plasmids. The application of this technique to Australian isolates of Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus), is discussed.
Identification of pathogenic yeasts of the imperfect genus Candida by polymerase chain reaction fingerprinting
Authors: Meyer Wieland , Latouche G. Nicolas , Daniel Heide‐Marie , Thanos Marcus , Mitchell Thomas G. , Yarrow David , Schönian Gabriele , Sorrell Tania C.
Published: May 2005

With the increase in the number of immunocompromised hosts, the number of fungal pathogens has increased markedly. Identification and classification, especially of yeast species and strains, is often difficult when based solely on phenotypic characteristics. Since it became clear that different fungal pathogens require specific treatment strategies, there is a need for simple, rapid and reliable methods to identify fungal isolates. Polymerase chain reaction (PCR) fingerprinting was successfully applied here to identify yeast isolates Microsatellite [(GTG)5; (GACA)4] and minisatellite [(5′GAGGGTGGCGGTTCT 3′), derived from the core‐sequence of the phage M13] specific primers were used as single primers in the PCR to amplify hypervariable interrepeat DNA sequences from over 200 European, American and Australian clinical isolates within the genus Candida. Each species, represented by its type strain, could be identified by a specific multilocus pattern, allowing for the assignment of all the isolates to the appropriate species. Intra‐species variation in the multilocus profiles was about 20% compared to inter‐species variation, which was up to 80%. Anamorph‐teleomorph pairs could be identified by highly homologous PCR fingerprint patterns. PCR fingerprinting was more discriminatory when compared with routinely used biochemical tests (Vitek YBC and API ID 32C). PCR fingerprinting has proven to be a powerful tool for the identification of medically important yeasts. It is rapid, sensitive, reliable, highly reproducible, stable in vitro and in vivo, and applicable to large‐scale experiments. Potential applications include: yeast taxonomy, epidemiology, environmental surveys, and improvement of the diagnosis of mycotic diseases.
Polymerase chain reaction‐linked single‐strand conformation polymorphism of ribosomal DNA to fingerprint parasites
Authors: Gasser Robin B. , Monti Jennifer R. , Zhu Xingquan , Chilton Neil B. , Hung Guo‐Chiuan , Guldberg Per
Published: May 2005

To overcome limitations of the morphological identification of some parasites (and their different developmental stages) to species, we have established a polymerase chain reaction‐linked single‐strand conformation polymorphism technique (PCR‐SSCP) utilizing the second internal transcribed spacer (ITS‐2) of ribosomal (r)DNA. This spacer was specifically chosen in the study of strongylid and ascarid nematodes because it is known to provide reliable species markers. The ITS‐2 from individual parasites was amplified by PCR, then denatured and subjected to electrophoresis on a mutation detection enhancement (MDE) (nondenaturing) gel matrix. PCR‐SSCP analysis showed that the single‐strand ITS‐2 patterns produced allowed the accurate identification of species. The method also allowed the display of (low‐level) variation in patterns within some species between different geographical isolates. These findings demonstrate the usefulness of PCR‐SSCP of ITS‐2 for the identification of nematode species and indicate its potential for resolving variation in the ITS‐2 sequence within species.
An evaluation of selected DNA extraction strategies for short tandem repeat typing
Authors: Vandenberg Nicholas , van Oorschot Roland. A. H. , Mitchell Robert J.
Published: May 2005

Organic and Chelex extraction methods are compared to five commercially available DNA extraction kits. For forensic purposes, comparison of different extraction strategies is based on the ability of the methods to economically recover high yields of amplifiable DNA from small blood samples in a quick, simple and safe manner. The findings of this study indicate that resin‐based methods, such as Chelex, are best able to meet these criteria.
Linkage analysis of bronchial hyperreactivity and atopy with chromosome 11q13
Authors: Wong Zilla Y. H. , Tsonis Dina , van Herwerden Lynne , Raven Joan , Forbes Andrew , Abramson Michael J. , Walters E. Haydn , Harrap Stephen B.
Published: May 2005

There are two key clinical features of asthma: allergy and bronchial hyperreactivity (BHR). Some pedigree studies of atopy have indicated linkage with the high affinity IgE receptor (FcϵRI‐β) gene on chromosome 11q13, but others failed to confirm this linkage. We examined the genetic linkage of three polymorphic microsatellite markers to atopy and BHR in 120 affected sibling pairs recruited from the general community. We found no linkage to atopy at any of the three 11q13 loci studied. Our findings also do not favour linkage between BHR and loci approximately 8–9 cM either side of the FcϵRI‐β gene.
Genetic analysis of the freshwater crayfish Cherax tenuimanus
Authors: Imgrund John , Groth David , Wetherall John
Published: May 2005

The marron (Cherax tenuimanus) is one of the few species of freshwater crayfish native to Australia that is suitable for aquaculture and occurs only in the southwest of Western Australia. This study describes polymorphic microsatellite markers which differentiate marron populations from several geographically distinct regions (including rivers and streams, dams, and commercial marron farms) throughout Western Australia. Twenty microsatellite loci, primarily of the (CA)n. (GT)n type, were isolated and sequenced from a marron cosmid library. Three of these loci were characterised further. Two loci exhibited extensive polymorphism and one was monomorphic. The polymorphic loci exhibited Mendelian codominant inheritance in the family group comprising two individual parents and approximately 100 offspring bred for this study. These loci permitted differentiation between the five geographically distinct populations studied and thus provide a basis for genetic characterisation of marron stock in Western Australia.
Minisatellite DNA markers reveal hybridisation between the endangered black robin and tomtit
Authors: Ma Wei , Lambert David
Published: May 2005

We have used multilocus minisatellite DNA profiling to assess genetic variation in the endangered and highly inbred black robins and the closely related tomtits. These species are restricted to wild populations on the Chatham Islands, off the east coast of New Zealand. DNA profiles revealed a remarkable degree of genetic similarity among black robins, as well as among the tomtits sampled, although the typical profiles of these two species were clearly distinct. The parentage of two putative hybrid chicks and one putative hybrid adult was established for a ‘black robin’ adult male and an adult tomtit female which were found nesting together. The ‘black robin’ male, despite being morphologically a typical black robin, was found to possess a number of minisatellite‐containing restriction fragments not recorded from the black robin population (n = 103). These fragments were however detected in tomtits. Consequently, this bird appeared to be an F1 hybrid and his three offspring were F2 hybrids. Mitochondrial DNA sequencing of members of this family showed that the ‘black robin’ male had cytochrome b and 16S of black robin, rather than tomtit, haplotypes. This indicates that the black robin adult male hybrid was the result of a black robin female mating with a tomtit male. These findings are particularly important since tomtits had been used as cross‐foster parents for black robins, as part of an earlier conservation recovery program The existence of any hybrids thus detected would have important implications for cross‐fostering programs generally, since may such programs are now being implemented in conservation programs worldwide.
Clinical potential of microchip capillary electrophoresis systems
Authors: Colyer Christa L. , Tang Thompson , Chiem Nqhia , Harrison D. Jed
Published: May 2005

Clinical interest in the use of capillary electrophoresis (CE) has recently been extended to the microchip environment. Clinical analyses demand careful handling of complex samples that are often limited in quantity and in concentration. The integrated sample handling and analysis capabilities of microchip substrates thus seem ideally suited to clinical applications. This review surveys the development of sample handling (injection, mixing, and reaction) and separation elements on‐chip. The integration of these elements to create a variety of clinical analyzers has been demonstrated. The application of microchip CE systems to human serum protein analysis, immunoassay, and DNA studies is reviewed, along with various other clinical applications. In addition, the clinical potential of the lab‐on‐a‐chip concept is discussed.
Comparison of serum protein electrophoresis by agarose gel and capillary zone electrophoresis in a clinical setting
Authors: Jolliff Carl R. , Blessum Cynthia R.
Published: May 2005

Capillary zone electrophoresis (CZE) offers the potential for automating serum protein electrophoretic analysis traditionally performed on standard thin‐layer agarose gels. The following describes the use of CZE compared to agarose gel electrophoresis (AGE) for the detection of dysproteinemia and paraproteinemia in a clinical study involving 240 patients. The study includes within‐run and between‐run reproducibility data on the Paragon CZE® 2000 Clinical Capillary Electrophoresis System, in addition to concordance data between the two methodologies. Paraprotein quantitation studies comparing AGE versus CZE were also performed. Reproducibility for the automated CZE system was superior to the AGE system. Improved reproducibility for the CZE method is largely due to measuring protein absorbance directly at 214 nm versus the traditional AGE method that measures the amount of dye adsorbed to protein. Reproducibility data as percent coefficient of variance (% CV) for the five classic bands in a normal control serum for between‐run precision ranged from 1.2 to 4.5% for CZE compared to AGE, which ranged from 3.8 to 8.0% CV. Concordance studies between AGE and CZE involving dysproteinemias including hypogammaglobulinemia, polyclonal and monoclonal gammopathies, acute and chronic inflammation, nephrosis, hepatodegenerative disease, cirrhosis, and iron deficiency anemia showed 96% agreement. Paraprotein classification, which compared the CZE immunosubtraction method to immunofixation electrophoresis (IFE) on agarose, showed 100% agreement. Certain dysproteinemias involving beta lipoprotein were in partial concordance due to the inability of the CZE procedure to detect this component. Detection limits for monoclonal gammopathies, providing they were not comigrating with other proteins, were IgG 50 mg/dL, IgM 75 mg/dL, and IgA 75 mg/dL. Paraprotein quantitative studies between the two methods showed less than a ± 0.2 g/dL variation.
Capillary isoelectric focusing of hemoglobin variants in the pediatric clinical laboratory
Authors: Hempe James M. , Granger Jerry N. , Craver Randall D.
Published: May 2005

We have used capillary isoelectric focusing (cIEF) to diagnose and monitor hemoglobinopathies in children for over three years. This report describes a major revision of our original method (Clin. Chem. 1994, 40, 2288–2295) that improves the analysis of hemoglobin (Hb) variants by cIEF, especially capillary performance and quantitation precision for minor variants. The revised method uses mixed ampholytes (2% pH 6‐8:3‐10; 10:1), lower viscosity methylcellulose (0.375%) solution, between‐sample capillary conditioning with methanol, and hemolysates prepared from red blood cells (RBC) instead of whole blood. Collectively, these changes prolonged capillary life by minimizing capillary exposure to NaOH, standardized the sample matrix, and improved the precision of peak autointegration. The between‐run quantitation imprecision (% relative standard deviation) of the revised method was 0.1–3.5% for all diagnostically important major and minor Hb variants present at normal or abnormal levels. The results show the use of the revised method for (i) post‐translationally modified Hb present at low concentrations in normal blood, (ii) Hb oxidation products produced by improper sample storage, (iii) differential diagnosis of S/β+ thalassemia, G‐Philadelphia trait, S/C‐Harlem disease, and Hb H disease, (iv) sensitive detection of minor variants like Hb A2′ as indicators of an α globin mutation, and (v) neonatal screening using dried blood collected on filter paper. The results show that high‐efficiency separation and precise quantitation of Hb variants over a wide range of concentrations makes cIEF a comprehensive assay that can be used without adjunct analyses for the automated primary evaluation of hemoglobinopathies and thalassemias.
Combination of solid‐phase extraction and field‐amplified concentration for trace analysis of organonitrogen pesticides by micellar electrokinetic chromatography
Authors: He Yan , Lee Hian K.
Published: May 2005

An analytical method based on the combination of solid‐phase extraction (SPE) and field‐amplified concentration (FAC), with micellar electrokinetic chromatography (MEKC) was developed to extract, concentrate, separate, and quantitate six organonitrogen pesticides (metribuzin, bromacil, terbacil, hexazione, triadimefon, and DEET) in drainage water. The recovery of the first five pesticides was over 85%, and the limit of detection was 0.8 ppb. Four advantages (enhanced enrichment and recoveries, reduced extraction time and interferences) of combining SPE with FAC for trace analysis of pesticides at the ppb level were demonstrated.
A novel negative imaging technique for accurate localization of stainable proteins on complex two‐dimensional autoradiograms
Authors: Naaby‐Hansen Soren , Roof Richard W. , Flickinger Charles J. , Grafer Constance M. , Parsons Sarah J. , Herr John C.
Published: May 2005

This paper describes a fast, simple, and accurate method for localization of protein antigens in complex two‐dimensional (2‐D) autoradiograms where the precise position and identity of the protein is required. The method involves the creation of negative images on an autoradiogram through arrangement of image‐intensifying screens. By placing a chromogenically stained 2‐D gel or blot between the intensifying screen and the film, photons emitted from the intensifying screen are obstructed by the stained spots, thus creating a negative image on the film. The technique can be used in autoradiograms of proteins labeled with either 32P or 125I radioisotopes. The technique permits analysis of radiolabeled, gold‐stained and immunoreacted proteins on a single film and offers versatility by combining analysis of total protein patterns with specific identification of radiolabeled and/or immunoreacted protein spots. The technique is especially useful when selecting a subset of specifically radiolabeled proteins from the total protein pattern in 2‐D gels or membranes for microsequencing.
Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two‐dimensional gel electrophoresis
Authors: Langen Hanno , Röder Daniel , Juranville Jean‐François , Fountoulakis Michael
Published: May 2005

Two‐dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5–200 kDa. For the first‐dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second‐dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.
Two‐dimensional electrophoretic analysis of the protein family at 90 kDa of abortifacientChlamydia psittaci
Authors: Giannikopoulou Panagiota , Bini Luca , Simitsek Phaedra D. , Pallini Vitaliano , Vretou Evangelia
Published: May 2005

Four major clusters, designated A, B, C and D, were distinguished in Western Blots by a monoclonal antibody specific for the “antigen family at 90 kDa” after two‐dimensional electrophoretic analysis on immobilized pH gradient of chlamydial elementary bodies of abortifacientC. psittaci. Clusters B, C, and D were closely related with molecular mass (kDa) pIvalues of 91.5/5.2–5.4, 90/5.0–5.2 and 90.5/5.6–5.8, respectively. Cluster A was larger, with molecular mass/pIof 104.7/5.1–5.3. Evidence for the antigenic relationship between cluster A and clusters B, C and D was further supported by immunological cross‐reaction with affinity‐purified antibodies from serum of ewes with chlamydial‐induced abortion. The experimental values obtained for size and pIof the four clusters correlated well with the calculated values from known sequences coded by multiple chlamydial genes. Direct evidence for the correspondence between the immunoreactive clusters B, C and D and the retrieved genes was provided by antibody binding experiments to recombinant polypeptides representing fragments of the deduced proteins. The 4‐member antigen family at 90/104 kDa is the first example of proteins coded by multiple genes within the genus Chlamydia.
DNA sequencing by capillary electrophoresis
Authors: Dovichi Norman J.
Published: May 2005

Capillary electrophoresis has been under development for DNA sequencing since 1990. This development has traveled down two parallel tracks. The first track studied the details of DNA separation by gel electrophoresis. Early work stressed rapid separations at high electric fields, which reached the extreme of a 3.5 min sequencing run at 1200 V/cm. While fast separations are useful in clinical resequencing applications for mutation detection, long read‐length is important in genomic sequencing. Unfortunately, sequence read‐length degrades as electric field and sequencing speed increases; this tradeoff between read‐length and sequencing speed appears to be a fundamental result of the physics of DNA separations in a polymer. The longest sequence sequencing read‐lengths have been obtained at modest electric fields, high temperature, and with low concentration noncrosslinked polymers. In parallel with our understanding of DNA separations, the second track of DNA sequencing development considered the design of large‐scale capillary instruments, wherein hundreds of DNA samples can be sequenced in parallel. Real‐world application of these very high throughput capillary electrophoresis systems will require significant investment in sample preparation technology.
Analysis of glycosaminoglycans and their oligosaccharide fragments by capillary electrophoresis
Authors: Grimshaw James
Published: May 2005

Glycosaminoglycans are high molecular mass polymers found in living tissues. They are degraded by site specific enzymes to oligosaccharide fragments. Polymers and oligomers all carry negative charge and are found as salts soluble in water. Consequently, capillary electrophoresis has come to the fore as an analytical technique in this field. This review discusses application of the technique to the chondroitin‐dermatan, heparin and hyaluronan groups of polymer and the derived oligosaccharides. Direct and indirect UV detection are discussed as well as detection methods involving precolumn derivatization. Capillary electrophoresis has found use in the characterisation of heparin and in following the changes in chondroitin and hyaluronan during the onset of arthritic disease in humans. Some glycosaminoglycans have been used as asymmetric anions in the separation of enantiomers of low molecular mass compounds by capillary electrophoresis.
Capillary electrophoresis of natural products
Authors: Issaq Haleem J.
Published: May 2005

Capillary electrophoresis (CE) and micellar electrokinetic chromatography were used for the separation of widely different compounds from natural materials including antibiotics, humic substances, flavonoids, isoflavonoids, illicit drugs, coumarins, alkaloids, steroids, Chinese herbal preparations, nicotine, caffeine, amphetamines, toxins such as aflatoxins B1, B2, G1, G2, mycotoxins, heptapeptide aflatoxins and others, ephedrine compounds, mineral elements, and natural compounds in biological samples. A discussion of sample extraction and clean‐up and the advantages of using CE is also presented.
Capillary electrophoresis in the determination of pollutants
Authors: Dabek‐Zlotorzynska Ewa
Published: May 2005

The applications of capillary electrophoresis to the analysis of environmental pollutants have been carefully revised from the literature compiled in the Current Contents and focused on the description of methods developed since 1994. The literature is reported according to the pollutant type. Groups of compunds which have received much attention include phenols, polynuclear hydrocarbons, amines, carbonyl compounds, surfactants, dyes and inorganic pollutants. Easy‐to‐read table provides useful information about the state‐of‐the‐art and possibilities offered by capillary electrophoresis (CE) in environmental analysis.
Analysis of subcellular organelles involved in major histocompatibility complex (MHC) class II‐restricted antigen presentation by electrophoresis
Authors: Engering Anneke , Lefkovits Ivan , Pieters Jean
Published: May 2005

Presentation of material derived from pathogenic organisms to the immune system requires uptake of antigens into antigen presenting cells, processing into peptide fragments and loading of the resulting fragments onto major histocompatibility complex (MHC) class II molecules. MHC class II‐restricted antigen presentation involves both the biosynthetic as well as the endocytic pathway of antigen‐presenting cells. In recent years, the general mechanisms that govern these processes have been delineated, and specialized organelles have been characterized in which processing and loading of antigens takes place. Here, we review the work that has led to the characterization of these MHC class II compartments, and describe the use of organelle electrophoresis and two‐dimensional gel electrophoresis to analyze the molecular composition of the different subcellular organelles involved in MHC class II‐restricted antigen presentation as well as in antigen uptake.
Two‐dimensional mapping of the endogenous proteins of the rat hepatocyte Golgi complex cleared of proteins in transit
Authors: Taylor Randall S. , Fialka Irene , Jones Steven M. , Huber Lukas A. , Howell Kathryn E.
Published: May 2005

The discovery of additional endogenous Golgi proteins will lead to significant new insights into Golgi function. To this end, stacked Golgi fractions (SGFs) were isolated from rat liver before (CTL SGF) and after molecules in transit through the Golgi were cleared by pre‐treatment with cycloheximide (CHX SGF). Electron microscopic (EM) morphometric and biochemical analyses showed that the in vivo stacked morphology is retained, that > 90% of the elements can be positively identified as Golgi stacks and cisternae, and that transmembrane protein markers of the Golgi complex are enriched 300‐ to 800‐fold over starting postnuclear supernatant (PNS) [24]. High‐resolution two‐dimensional (2‐D) gel mapping has been carried out on the CTL PNS, CTL SII (an intermediate fraction), CTL SGF, CHX SGF, CHX SGF – high pH supernatant, and CHX SGF – high pH pellet. This analysis, coupled with immunoblotting and alignment of the 2‐D gels with master gels, has allowed the identification of a number of known proteins and the preliminary characterization of the most abundant 173 Golgi‐specific proteins. These 173 proteins have been placed into three categories: cargo, cytosolic Golgi‐associated, and resident Golgi proteins. These categories are tentative and will be modified as more data are acquired from immunoblotting and protein sequencing.
Biogenesis of lipoprotein(a) in human and animal hepatocytes
Authors: Lobentanz Eva‐Maria , Dieplinger Hans
Published: May 2005

The atherogenic plasma lipoprotein complex Lp(a) consists of low density lipoprotein (LDL) and the highly polymorphic glycoprotein apolipoprotein(a) covalently linked by a disulfide bridge. A size polymorphism of apolipoprotein(a) results from a variable number of tandemly arranged kringle IV repeats. The largely varying plasma concentration of Lp(a) is nonnormally distributed in the population and correlates inversely with the molecular mass of apolipoprotein(a). In vivo turnover studies have revealed that differences in Lp(a) plasma concentrations reflect different synthesis rather than degradation. Plasma Lp(a) originates exclusively in the liver. Detailed studies of the intracellular metabolism of apolipoprotein(a) in transfected human hepatoma cells as well as in primary baboon hepatocytes have revealed an unusual secretory pathway of this protein. Due to complex folding and processing, an immature precursor form of apolipoprotein(a) is retained in the endoplasmic reticulum for a prolonged time. This retention leads to a massive accumulation in the endoplasmic reticulum which stands in contrast to most secretory proteins. Since the retention time correlates positively with the apolipoprotein(a) isoform size, this intracellular mechanism could explain the inverse correlation between the isoform size and plasma concentrations observed in the general population. These findings therefore demonstrate a novel cellular regulatory mechanism for a secretory human plasma protein with genetically controlled concentrations. The majority of the above‐mentioned studies revealed another unusual feature of the biogenesis of Lp(a). The mature Lp(a) complex is formed, at least in the investigated cell models, only following separate secretion of apolipoprotein(a) and LDL‐like particles. Work that is related to both aspects of Lp(a) formation, both from our laboratory and from other authors, is reviewed.
SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling
Authors: Guex Nicolas , Peitsch Manuel C.
Published: May 2005

Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, we have developed an environment for comparative protein modeling that consists of SWISS‐MODEL, a server for automated comparative protein modeling and of the SWISS‐PdbViewer, a sequence to structure workbench. The Swiss‐PdbViewer not only acts as a client for SWISS‐MODEL, but also provides a large selection of structure analysis and display tools. In addition, we provide the SWISS‐MODEL Repository, a database containing more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, we hope to increase the use of protein structures and models in the process of experiment design.
The 2DWG meta‐database of two‐dimensional electrophoretic gel images on the Internet
Authors: Lemkin Peter F.
Published: May 2005

The 2DWG meta‐database is a searchable database of two‐dimensional (2‐D) electrophoretic gel images found on the Internet. A meta‐database contains information about locating data in other databases – but not that data itself. This database was constructed because of a need for an enriched set of World Wide Web (WWW) locations (URLs) of 2‐D gel images on the Internet. These gel images are used in conjunction with the National Cancer Institute (NCI) Flicker Server to manipulate and visually compare 2‐D gel images across the Internet. User's gels may also be compared with those in the database. The 2DWG is organized as a spreadsheet table with each gel image being represented by a row sorted by tissue type. Data for each gel includes tissue type, species, cell‐line, image URL, database URL, gel protocol, organization URL, image properties, map URL if it exists, etc. The 2DWG may be searched to find relevant subsets of gels. Searching is done using the dbEngine – a WWW database search engine which accesses selected rows of gels from the full 2DWG table. The 2DWG meta‐database is accessible on the WWW at http://www‐lecb.ncifcrf.gov/2dwgDB/ and the NCI Flicker server at http://www‐lecb.ncifcrf.gov/flicker/.
Variability in the pattern of random amplified polymorphic DNA
Authors: Khandka Deepak K. , Tuna Metin , Tal Moshe , Nejidat Ali , Golan‐Goldhirsh Avi
Published: May 2005

The random amplified polymorphic DNA (RAPD) technique is a simple method to detect DNA polymorphism. It is sensitive to reaction conditions. Small changes in the reactants' concentration cause variations in amplification products. Using DNA from Asparagus officianalis, Dactylis glomerata, Mercurialis annua and Escherichia coli, we examined variability in the amplification pattern associated with reaction constituents. An increase in the ratio of Taq DNA polymerase to DNA in the reaction increased the number of amplified fragments. Increasing the concentration of primer resulted in the amplification of low molecular weight DNA fragments, while lowering the concentration resulted in high molecular weight fragments. Subsets of amplified fragments required different concentrations of magnesium for their highest intensity. Mechanical shearing of DNA obtained by sonication led to reduction in amplification of a subset of products. Enzymatic fragmentation of DNA by restriction enzymes led to loss or gain of specific fragments, depending on the DNA, primer, and restriction enzyme. RAPD markers of pooled DNA of anonymous pedigree should be critically evaluated for frequent ‘false positive’ markers.
Characterization of dye‐induced mobility shifts affecting DNA sequencing in poly(ethylene oxide) sieving matrix
Authors: Tan Hongdong , Yeung Edward S.
Published: May 2005

The influence of three classes of fluorescence labels including dipyrrometheneboron difluoride (BODIPY), energy transfer (ET) and conventional fluorescein and rhodamine (ABI), on DNA sequencing has been examined with laser‐induced fluorescence detection and poly(ethylene oxide)‐filled capillary electrophoresis. DNA sequencing fragments were generated by dye‐labeled primer cycle‐sequencing reactions in a hot‐air thermal cycler. A parameter, relative‐induced shift, was introduced to quantify the uniformity of electrophoretic mobilities of these fragments. BODIPY was found to have the smallest, but nonzero, effect for dye‐induced nonuniformity. Although ET dyes provided the highest sensitivity due to their unique spectroscopic properties, they were found to lack photostability compared to BODIPY and ABI dyes. Characterization also brings out some important tips for selecting the suitable dye set for the two‐channel ratio‐based DNA base‐calling method.
Detection of p53 point mutations by double‐gradient, denaturing gradient gel electrophoresis
Authors: Gelfi Cecilia , Righetti Sabina C. , Zunino Franco , Della Torre Gabriella , Pierotti Marco A. , Righetti Pier Giorgio
Published: May 2005

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30–80% or 40–80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5–12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single‐strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.