bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capillary isoelectric focusing of hemoglobin variants in the pediatric clinical laboratory
Authors: Hempe James M. , Granger Jerry N. , Craver Randall D. 
Published: May 2005

AbstractWe 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

AbstractAn 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

AbstractThis 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

AbstractTwo‐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

AbstractFour 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

AbstractCapillary 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

AbstractGlycosaminoglycans 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

AbstractCapillary 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

AbstractThe 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

AbstractPresentation 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

AbstractThe 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

AbstractThe 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

AbstractComparative 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractGenetic 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.