bluuebunny/crossref_metadata_2025_split huggingface dataset:
D12S67, a bipartite locus: Differential amplification of parts of the nucleotide sequence reveals considerable polymorphism
Authors: Minaguchi Kiyoshi 
Published: May 2005

AbstractThe 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

AbstractTwo 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

AbstractIn 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

AbstractThe 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

AbstractThe 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

AbstractNoncross‐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

AbstractCapillary 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

AbstractA 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

AbstractPesticides 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

AbstractFollowing 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

AbstractPolarized 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

AbstractElectrophoretic 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

AbstractWhole 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

AbstractAn 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

AbstractA 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

AbstractAn 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

AbstractA 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

AbstractConventional 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

AbstractThe 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.