bluuebunny/crossref_metadata_2025_split huggingface dataset:
The use of mixed surfactants in micellar electrokinetic chromatography
Authors: Ong Chye P. , Ng Choon L. , Lee Hian K. , Li Sam F. Y. 
Published: May 2005

AbstractThe use of mixed surfactants for the separation of nine phenylthiohydantoin amino acids (PTH amino acids) by micellar electrokinetic chromatography (MEKC) was examined. The potential of a mixed surfactant system, consisting of cationic and anionic surfactants, for enhancing the selectivity of MEKC separations was explored. Both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) in the electrophoretic medium were found to improve the separation of the PTH‐amino acids compared to the use of CTAB or SDS alone. Complete separation of the PTH amino acids was achieved using a buffer containing 2.5 mM CTAB and 40 mM SDS. The analysis was performed without the need to reverse the polarity of the electrodes, typically required if oppositely charged micelles are used. The migration behavior of the nine PTH amino acids using the mixed surfactant system was compared with MEKC in presence of SDS alone.
Micellar electrokinetic chromatographic studies in D2O based buffer solutions
Authors: Greenaway Mark , Okafo George , Manallack David , Camilleri Patrick 
Published: May 2005

AbstractThe addition of deuterium oxide or organic solvents to surfactant‐based electrolytes is an effective method for improving resolution in micellar electrokinetic capillary chromatography (MECC). Buffers containing phosphate, boric acid and sodium dodecyl sulphate at pH or pD 7 in the presence and absence of deuterated and non‐deuterated solvents were used to separate some hydrophobic compounds covering a range of octanol‐water partition coefficients (log P). Significant improvements in resolution of the more hydrophobic compounds were achieved by using mixtures of heavy water and deuterated methanol, instead of water and methanol. Quantitative structure‐retention relationships were also derived to provide a better understanding of the intermolecular interactions that contribute to the separation of analytes in MECC. Multiple regression analysis has shown that capacity factors were significantly related to both log P and molecular refractivity. The latter is a measure of the molecular volume of analytes and is related to their ability to undergo dispersive interactions with the micellar components.
Micellar capillary electrophoresis separation and thermo‐optical absorbance detection of products from manual peptide sequencing
Authors: Chen Min , Waldron Karen C. , Zhao Yuwei , Dovichi Norman J. 
Published: May 2005

AbstractMicellar capillary electrophoresis is optimized for separation of phenylthiohydantoin (PTH) amino acids produced in manual Edman degradation reaction for protein sequencing. There are also two major side‐products produced by the Edman degradation reaction: diphenylthiourea and dimethylphenylthiourea. We report the complete separation of 19 PTH amino acids plus the two major side‐reaction products in 10 min. Capillary electrophoresis is used to identify the five residues generated by manual Edman degradation sequencing of a pentapeptide.
Micellar electrokinetic chromatography of the anti‐human immunodeficiency virus (HIV) agent michellamine B with absorption and laser‐induced fluorescence detection
Authors: Chan King C. , Majadly Fathy , McCloud Thomas G. , Muschik Gary M. , Issaq Haleem J. , Snader Kenneth M. 
Published: May 2005

AbstractMicellar electrokinetic chromatography (MEKC) was applied to the separation of the anti‐HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium de‐oxycholate and 10–20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high‐performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser‐induced fluorescence detection (LIF). Using the sodium dodecyl sulfate‐containing buffer and LIF, the limit of detection for michellamine B was ∼ 2 ng/mL. The sensitivity was degraded ∼100‐fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.
The human myocardial two‐dimensional gel protein database: Update 1994
Authors: Corbett Joseph M. , Wheeler Colin H. , Baker Cathy S. , Yacoub Magdi H. , Dunn Michael J. 
Published: May 2005

AbstractAn updated human heart protein two‐dimensional electrophoresis (2‐DE) database is presented. The database, which contains some 1388 protein spots characterised in terms ofMrand pI, has been analysed further by Western immunoblotting and protein sequencing. From a total of 103 protein spots analysed, 49 have been identified by immunoblotting and 32 have been identified by protein sequencing. A further six proteins have tentatively been assigned by comparison with the human heart 2‐DE protein database of Jungblutet al. (Electrophoresis) 1994,15, 685–607). This database is being used in studies of alterations in protein expression in the diseased and transplanted human heart.
Electric birefringence imaging of DNA in agarose electrophoresis gels: Noise limits and prolonged electric field effects
Authors: Lanan Maureen , Grossmann Daniel W. , Morris Michael D. 
Published: May 2005

AbstractElectric birefringence imaging (EBI) maps stresses in agarose electrophoresis gels. Both DNA and gel alignment can be monitored using this technique. Previously, we have reported birefringence images associated with resolved small (< 48 kbp) and partially resolved large 48 kb – (2 Mbp) DNA fragments oriented with relatively short electric field pulses. In this paper, differences between images acquired using short and long (DC) voltage pulses are presented. It is shown that stress signals distribute over long distances when exposed to prolonged electric fields. Resolution also affects band shapes. Refinements in instrumental design and evaluation of limiting noise sources of the technique are also described.
An algorithm for unwarping multitrack electrophoretic gels
Authors: Glasbey Chris A. , Wright Frank 
Published: May 2005

AbstractAn algorithm is proposed for removing the effect of “frowns” and “smiles” (i.e. warping) from multitrack electrophoretic gels, so that the bands are horizontal and in alignment between tracks. It has been applied to a DNA sequencing autoradiograph, but could equally be applied to other types of gel separation where warping occurs. The algorithm uses a gradient filter to estimate band orientations. These are smoothed and interpolated by robust regression, and integrated to find the unwarping transform. No manual intervention is required.
Calculation of paternity probabilities from multilocus DNA profiles
Authors: Brenner Charles H. , Rittner Christian , Schneider Peter M. 
Published: May 2005

AbstractWe describe a procedure for evaluation of paternity evidence from multi‐locus DNA probe patterns. A computer program abstracts a “+/−” notation description from the multilocus profile and then calculates a paternity index based on observed phenotypic fragment frequencies. The biostatistical evaluation considers only bands found in the child and missing from the mother ‐ a simplified approach that is at once robust and conservative. Mutations are of course taken into account. Particular features lending objectivity to the interpretation include computer reading and matching decisions, and specific recognition and statistical compensation for ambiguities (“faint orphans”).
Towards new formulations for polyacrylamide matrices: N‐acryloylaminoethoxyethanol, a novel monomer combining high hydrophilicity with extreme hydrolytic stability
Authors: Chiari Marcella , Micheletti Claudia , Nesi Marina , Fazio Marco , Righetti Pier Giorgio 
Published: May 2005

AbstractMatrices for electrokinetic separations, based on a unique class of mono‐ and disubstituted (on the amido nitrogen) acrylamides such as e.g., N‐acryloylaminoethoxyethanol (AAEE) and acrylamido‐N,N‐diethoxyethanol, offer the following advantages: (i) strong resistance to alkaline hydrolysis (most zone separations occurring at basic pH values), (ii) high hydrophilicity and (iii) greater porosity, due to the higher molecular weight of the monomers. When compared with conventional poly(acrylamide), a poly(AAEE) matrix, when subjected to mild alkaline hydrolysis (0.1 N NaOH, 70°C) appears to be 500 times more stable. Such stability is also confirmed under strong alkaline hydrolysis (1 N NaOH, 100°C) as well as under mild and strong acidic hydrolysis. Mildly hydrolyzed poly(AAEE) matrices still perform extremely well in both conventional isoelectric focusing and immobilized pH gradients, techniques which are quite sensitive to traces of acrylate in the polymer coil. Conversely, mildly hydrolyzed poly(acrylamide) matrices, when used in isoelectric focusing, generate pH gradients between pH 4 and 5, having an inflection point (pH 4.6) equivalent to the pK value of acrylic acid. This novel class of monomers shows great promise for future applications in all electrokinetic methodologies.
Differential detergent fractionation of isolated hepatocytes: Biochemical, immunochemical and two‐dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments
Authors: Ramsby Melinda L. , Makowski Gregory S. , Khairallah Edward A. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment‐ or stage‐specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergentcontaining buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane‐organelle, nuclear membrane and cytoskeletal‐matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat‐shock 70 proteins, glutathione‐S‐transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2‐D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2‐D gel electrophoretic analysis. Extensive 2‐D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of Mr and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2‐D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).
Two‐dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte‐pH gradients
Authors: Rabilloud Thierry 
Published: May 2005

AbstractA modified procedure for the two‐dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate‐electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained.
Mapping small GTP‐binding proteins on high‐resolution two‐dimensional gels by a combination of GTP binding and labeling with in situ periodateoxidized GTP
Authors: Huber Lukas A. , Peter Marcus E. 
Published: May 2005

AbstractWe compared two approaches to identify and map small GTP‐binding proteins in combination with high‐resolution two‐dimensional (2‐D) gel electrophoresis. The first approach involved direct GTP ligand binding after a renaturing transfer onto nitrocellulose. In the second, affinity labeling with in situ periodate‐oxidized GTP was used in permeabilized cells (Peter, M. E., She, J., Huber, L. A. and Terhorst, C. Anal. Biochem. 1993, 210, 77–85). Analysis by 2‐D gel electrophoresis revealed a number of distinct intracellular small GTP‐binding proteins in Madine‐darby canine kidney strain II cells (MDCKII). Using specific antibodies the electrophoretic coordinates of rab4, rap1a/b, and rap2 were identified for native as well as for crosslinked GTPases. These methods allow the identification of small GTP‐binding proteins in total cell lysates and purified subcellular fractions, providing excellent markers throughout the course of differentiation and development.
Discovery and characterization of two novel human cancer‐related proteins using two‐dimensional gel electrophoresis
Authors: Leavitt John 
Published: May 2005

AbstractComparative examination of protein synthesis in normal and neoplastic human fibroblasts led to the discovery of two novel microfilament proteins with roles in human neoplasia. One protein, a mutant β‐actin was found to convert nontumorigenic human fibroblasts to tumorigenicity. Recently, the oncogenic potential of this mutant β‐actin was verified independently and shown to alter the metastatic phenotype of human cells in conjunction with the myc and ras oncogenes. A second protein, leukocyte plastin, was discovered to be a marker of a majority of human cancer cells of nonhemopoietic origin. A survey of SV40‐transformed human fibroblasts and human sarcoma and carcinoma cell types demonstrated that the L‐plastin gene was activated at widely varying degrees in nearly all human cancer cells. Activation of the L‐plastin gene was not detected in normal nonhemopoietic cells using sensitive reverse transcript‐polymerase chain reaction, excepting those cells that expressed estrogen and progesterone receptors which mediate activation of L‐plastin synthesis in reproductive tissues. Our most recent findings have revealed that activation of L‐plastin synthesis in neoplastic cells that cannot phosphorylate L‐plastin (e.g. those neoplastic cell types that express only trace amounts of L‐plastin) results in the coinduction of two alternative inflammatory programs of gene expression which mediate cytolytic effects on surrounding cells. This inflammatory response appears to be mediated by “inappropriate” constitutive synthesis of L‐plastin and failure of the induced cell to phosphorylate L‐plastin. Our findings suggest explanations for the novel resistance of human cells to in vitro transformation and one role of oncogene activation in cancer. As a consequence of the interplay of two‐dimensional (2‐D) gel electrophoretic analyses with other sophisticated techniques of molecular biology, the formal characterization of two fundamentally important multigene families was completed with determination of many aspects of the structure and function of these proteins and their genes. The discovery and characterization of the mutant β‐actin and L‐plastin and their relationship to the human neoplastic phenotype serve as useful models for the discovery of other important disease‐related proteins/genes using 2‐D gel electrophoresis.
Expression of tumor‐associated protein variants in chemically induced rat hepatomas and transformed rat liver cell lines determined by two‐dimensional electrophoresis
Authors: Zeindl‐Eberhart Evelyn , Jungblut Peter , Rabes Hartmut M. 
Published: May 2005

AbstractHigh resolution two‐dimensional electrophoresis (2‐DE), using the rat liver as a model, was applied to study hepatocarcinogenesis induced by different chemical carcinogens. Several tumor‐associated protein variants were detected by 2‐DE in chemically induced rat hepatomas and transformed rat liver cell lines compared to normal rat liver tissue. Proliferation‐related protein changes and/or protein alterations due to culture conditions were corrected for by comparison with 2‐DE patterns of isolated cells from regenerating liver and short‐term cultivated liver cells. Some of the tumor‐associated variants were further characterized: (i) By peptide mapping with limited proteolysis we detected clear relationships between several variants. (ii) By studying posttranslational modifications phosphorylated and glycosylated variants could be determined. (iii) A tumor‐associated protein variant was identified by amino acid analysis and amino acid sequencing.
Studying the interaction of polyoma virus middle T antigen with cellular proteins
Authors: Krauzewicz Nina S. 
Published: May 2005

AbstractThe oncogenic mouse polyoma virus encodes six proteins, two of which (the large and middle T antigens), when expressed together in primary rodent cells, generate an alteration of growth patterns commonly known as cellular transformation. The transformed cells grow with an unlimited life span and when introduced into immunocompromised mice rapidly form tumours. The large T gene product confers an indefinite growth potential on primary cells; however, the middle T gene product has been identified as bringing about the changes which ultimately allow the cell to form tumours. The 55 kDa middle T antigen has been shown to associate with a number of cellular enzymes involved in regulation of growth factor signalling pathways, all of which were identified as being components of the immunocomplexes that can be isolated from transformed cells using middle T antigen specific antibodies. Two‐dimensional gels have assisted the search for less prominent species present in these complexes. These methods represents one approach to investigating associating proteins, and as such, select for those interactions that are stable under the conditions used. In order to explore the possibility that middle T antigen could form complexes with other cellular proteins given different conditions, recombinant middle T antigen was used in a series of “filter overlay” experiments.
Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin‐layer cellulose plates
Authors: van der Geer Peter , Hunter Tony 
Published: May 2005

AbstractIdentification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P‐labeled proteins with site‐specific proteases and separation of the digestion products in two dimensions on thin‐layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P‐disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two‐dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin‐layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site‐specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two‐dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.
A rapid method for detection of N‐acetylglucosaminidase‐type chitinase activity in crossed immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels using 4‐methylumbelliferyl‐N‐acetyl‐D‐glucosaminide as substrate
Authors: Chen Kun‐Shang , Lee Kuo‐Kau , Chen Hsing‐Chen 
Published: May 2005

AbstractA method for the detection of N‐acetylglucosaminidase (GlcNAcase) activity has been developed by using 4‐methyl‐umbelliferyl‐N‐acetyl‐D‐glucosaminide (4‐MU‐GlcNAc) as substrate in crossed immunoelectrophoresis (CIE) and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Visualization of the reaction with a 366 nm ultra‐violet light is possible in approximately 30 min. The method is fast and sensitive in comparison with previous methods. The same band as in SDS‐PAGE, showing both GlcNAcase and chitinase activity, was found in the present study; we therefore conclude that this method is also useful in a GlcNAcase‐type chitinase assay.
The homology of a novel polypeptide with stress protein characteristics in embryonic mice brain and in the hypothalamus of caloric restricted rats as determined by ultramicro western blotting
Authors: Pipkin James L. , Hinson William G. , Lyn‐Cook Lascelles E. , La Borde James , Anson Jeanne F. , Feuers Ritchie J. , Duffy Peter H. , Hart Ronald W. , Casciano Daniel A. 
Published: May 2005

AbstractA novel protein (p34) was observed in polyacrylamide gel fluorographs of gestation day 13 embryonic mouse brain following retinoic acid dosing of dams. Another p34 polypeptide with identical gel migratory characteristics was seen in the hypothalamus of old caloric restricted rats after “food deprivation stress”. Western blotting, employing an ultramicro trans‐blot cell developed in our laboratory, detected identical immunochemical determinants between these proteins, verifying their homology. Peptide mapping and Western blotting further validated the uniqueness of p34 compared with other stress proteins including heme oxygenase.
Alzheimer brain proteins investigated by two‐dimensional gel electrophoresis with immobilized pH gradients in the first dimension
Authors: Mattila Kari M. , Frey Harry 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis with immobilized pH gradient 4–8 in the first dimension was applied in the analysis of Alzheimer's disease brain proteins. The silver‐stained 2‐D maps of extracts from the frontal cerebral cortex were examined. About 800 and 550 protein spots could be observed on the electrophoretograms from the total and buffer‐soluble fractions, respectively. In comparing the gels, four protein spots could be seen in all Alzheimer 2‐D patterns which were either undetectable (one spot) or significantly weaker (two spots) or stronger (one spot) in density than those from the controls.