bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional map of basic proteins of Haemophilus influenzae
Authors: Fountoulakis Michael , Takács Béla , Langen Hanno 
Published: May 2005

AbstractHaemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two‐dimensional gel electrophoresis, during which immobilized pH 3–10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two‐dimensional gel electrophoresis, using for the first‐dimensional separation immobilized pH gradient strips comprising the pH region of 6–11. The protein spots were analyzed by matrix‐assisted laser desorption ionization‐mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two‐dimensional map.
Two‐dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice
Authors: Kaino Seiji , Furui Toshifumi , Hatano Satoko , Kaino Miyuki , Okita Kiwamu , Nakamura Kazuyuki 
Published: May 2005

AbstractProteolytic enzymes in human pure pancreatic juice (PPJ), which was collected by cannulating the main pancreatic duct using endoscopy, were investigated by two‐dimensional zymography (2‐DZ). 2‐DZ was carried out by combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the second dimension, using gels containing casein or gelatin as a substrate for the proteolytic enzymes. After electrophoresis, the gels were incubated in Triton X‐100 followed by incubation at 37°C in Tris buffer (pH 8.5) containing CaCl2. By staining the gels with Coomassie Brilliant Blue (CBB R‐250), proteolytic enzymes were detected as clear spots and zones against a blue background. Proteinase inhibitors, such as a cysteine proteinase inhibitor (E‐64), a metalloproteinase inhibitor (EDTA), and a serine proteinase inhibitor (Pefabloc® SC), were added to PPJ in order to determine the types of proteinases. In patients with pancreatic cancer, spots of molecular weight (Mr) 70 000 and isoelectric points (pI) 5.3–5.5 were clearly detected on the gels containing casein and gelatin, while these spots were not detected in the PPJ from healthy subjects. The proteolytic activities of these spots were strongly inhibited by EDTA and Pefabloc SC but not E‐64. These results suggest that the spots of Mr 70 000 and pI 5.3–5.5 in PPJ of pancreatic cancer might be matrix metalloproteinase 2, which is a candidate for tumor‐associated proteinase. 2‐DZ proved to be a tool for analysis of proteolytic enzymes in PPJ and for the clinical diagnosis of pancreatic cancer.
Two‐dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins
Authors: Mortarino Michele , Tedeschi Gabriella , Negri Armando , Ceciliani Fabrizio , Gottardi Luciano , Maffeo Giovanni , Ronchi Severino 
Published: May 2005

AbstractA two‐dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N‐terminally sequenced. Major proteins already described in bull seminal plasma, like PDC‐109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N‐terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.
Development of a two‐dimensional gel electrophoresis database of human lysosomal proteins
Authors: Chataway Timothy K. , Whittle Alison M. , Lewis Martin D. , Bindloss Colleen A. , Moritz Robert L. , Simpson Richard J. , Hopwood John J. , Meikle Peter J. 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis databases have been generated for a range of tissue cell and fluid types, from a number of species. A major difficulty in the development of such databases is the large number of proteins present in even a single cell type (> 10 000) and the low levels of many of these proteins. One approach to overcome these difficulties is to fractionate the cell into its organelles and generate individual databases for each subcellular component. This has the added advantage of assigning a cellular location to each protein identified. Here we report the development of a two‐dimensional gel electrophoresis database of lysosomal proteins.
Additional possible tools for identification of proteins on one‐ or two‐dimensional electrophoresis
Authors: Tsugita Akira , Kamo Masaharu , Miyazaki Kenji , Takayama Mitsuo , Kawakami Takao , Shen Ruqun , Nozawa Takehiro 
Published: May 2005

AbstractAdditional, essentially chemical, identification methods of proteins in polyacry‐lamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C‐side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90° for 4–16 h, and the N‐side of serine/threonine with an S‐ethyl trifluorothioacetate vapor at 50° for 6–24 h. The products were analyzed by mass spectrometry– peptide mass fingerprinting. A new type of C‐terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90° for 2–16 h provided cleavages at the C‐side of aspartic acid and the N‐side of serine/threonine and simultaneous successive truncation at the C‐termini of the cleaved fragments. The product resulted in C‐terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at–5° for 8 h deblocked the N‐formyl group, and the vapor at 20° for 4 h deblocked pyrrolidone carboxylate. N‐acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50° for 1 h, followed by reaction with p‐suifophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C‐terminal sequencing of protein from polyacrylamide gels is also described.
Analyzing glycoproteins separated by two‐dimensional gel electrophoresis
Authors: Packer Nicolle H. , Lawson Margaret A. , Jardine Daniel R. , Sanchez J.‐C. , Gooley Andrew A. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as ‘trains’ of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the ‘rules’ which govern the migration of glycoproteins in 2‐D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one‐dimensional (1‐D) and 2‐D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N‐linked oligosaccharides) or chemically (β‐elimination of O‐linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC‐PAD) and identified by electrospray ionization mass spectrometry (ESI‐MS) or analyzed directly by ESI‐MS. After enzymic removal of the N‐linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O‐linked oligosaccharides. These approaches have been proved on 1‐D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2‐D PAGE preparative narrow range (pH 4.5–5.5) blots of human plasma: α2‐HS glycoprotein (human fetuin) and α1‐antitrypsin (α1‐protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2‐D PAGE.
Identification of phosphorylated proteins from thrombin‐activated human platelets isolated by two‐dimensional gel electrophoresis by electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS) and liquid chromatography‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS)
Authors: Immler Dorian , Gremm Dagmar , Kirsch Dieter , Spengler Bernhard , Presek Peter , Meyer Helmut E. 
Published: May 2005

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a powerful tool to separate complex protein mixtures including whole cell lysates. In combination with immunoblotting techniques or radioactive labeling techniques it is a fast and convenient way to demonstrate the presence of certain proteins or protein modifications. With the development of extremely sensitive analytical techniques such as matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) or electrospray ionization (ESI)‐MS, it has become possible to use 2‐D gels not only as an analytical but also as a preparative tool. Starting with a number of spots excised from 2‐D gels, a protein can be identified using different strategies involving enzymatic cleavage of the protein in the gel matrix, elution of the resulting peptides and analysis of these peptides by mass spectrometry. The obtained peptide mass fingerprint or fragment ion spectra from peptides can be used to screen protein or nucleic acid databases in order to identify the protein. We have used the techniques described above to identify proteins from human platelets which change their phosphorylation state following activation of platelets by thrombin. Platelets were radioactively labeled with [32P]orthophosphate and stimulated. Several protein spots in the observed range of 10–80 kDa and an isoelectric point of 3–10 showed a significant increase or decrease in phosphorylation. We present the results from the investigation of a spot group representing different isoforms and phosphorylation states of myosin light chain.
An isoelectrically trapped enzyme reactor operating in an electric field
Authors: Righetti Pier Giorgio , Bossi Allessandra 
Published: May 2005

AbstractMembrane enzyme reactors constitute an attempt at integrating catalytic conversion, product separation and/or concentration and catalyst recovery into a single operation. Whereas conventional membrane reactors confine an enzyme, in a free form, to one side of a membrane by size exclusion, electrostatic repulsion, or physical or chemical immobilization onto an intermediate support (gel, liposome), the membrane reactor here described is shown to operate under an entirely new principle: enzyme confinement into an isoelectric trap located in a multicompartment electrolyzer operating in an electric field. Two isoelectric membranes, having pIvalues encompassing both the enzyme pIand the pH of its optimum of activity, act by continuously titrating the enzyme trapped inside, thus preventing it from escaping the reaction chamber. Charged products generated by the enzyme catalysis are continuously electrophoretically transported away from the reaction chamber and collected into other chambers stacked either towards the cathodic or anodic sides. In a urease reactor, ammonia is continuously harvested towards the cathode, thus allowing >95% substrate consumption with maintenance of enzyme integrity over much longer time periods than in a batch reactor. In a trypsin reactor, casein is digested and biologically active peptides are continuously harvested in a pure form into appropriate isoelectric traps. In a third example, pureD‐phenylglycine is produced from a racemate mixture, via an acylation reaction onto a cosubstrate (the ester methyl‐4‐hydroxyphenyl acetate), brought about by the enzyme penicillin G acylase.
Counterbalancing hydrodynamic sample distortion effects increases resolution of free‐flow zone electrophoresis
Authors: Weber Gerhard , Bauer Johann 
Published: May 2005

AbstractOn fractionation of highly heterogeneous protein mixtures, optimal resolution was achieved by forcing proteins to migrate through a preestablished pH gradient, until they entered a medium with a pH similar but not equal to their pIs. For this purpose, up to seven different media were pumped through the electrophoresis chamber so that they were flowing adjacently to each other, forming a pH gradient declining stepwise from the cathode to the anode. This gradient had a sufficiently strong band‐focusing effect to counterbalance sample distortion effects of the flowing medium as proteins approached their isoelectric medium closer than 0.5 pH units. Continuous free‐flow zone electrophoresis (FFZE) with high throughput capability was applicable if proteins did not precipitate or aggregate in these media. If components of heterogeneous protein mixtures had already started to precipitate or aggregate, in a medium with a pH exceeding their pI by more than 0.5 pH units, the application of interval modus and media forming flat pH gradients appeared advantageous.
Analysis of trypanosomal endocytic organelles using preparative free‐flow electrophoresis
Authors: Grab Dennis J. , Webster Paul , Lonsdale‐Eccles John D. 
Published: May 2005

AbstractIn this paper we demonstrate the power of preparative free‐flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free‐flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time‐dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conlusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket‐derived membranes from other endosomal and lysosomal compartments of African trypanosomes.
Characterization of a subpopulation of mouse red blood cells as preferential target for malarial invasion
Authors: Sabolovic Domagoj , Sestier Claude 
Published: May 2005

AbstractCell electrophoresis was used to study a distinct subpopulation of murine red blood cells (RBC). These RBC are normally found in the spleen and bone marrow. They appear in the peripheral blood when mice are mildly bled or sucked by mosquitos. These cells have been characterized as having larger size, light density, lower electrophoretic mobility, and being more resistant to lysis with unsaturated fatty acids and in glycerol‐containing medium than mature erythrocytes. All their features suggest that their differentiation status represents an intermediate stage between reticulocytes and adult RBC. In vitro Plasmodium invasion tests showed their increased sensitivity to invasion by the parasites. The extent of their spreading in the blood was found to be strain‐dependent, being more pronounced in C57B1/6 mice, highly susceptible to developing cerebral malaria after infection with Plasmodium berghei, as compared to Balb/c, a nonsusceptible strain of mice.
Capillary electrophoretic analysis of ginseng polypeptide
Authors: Kajiwara Hideyuki , Hemmings Andrew M. 
Published: May 2005

AbstractA ginseng polypeptide (GPP) found in ginseng roots and its modified peptides were tested by capillary zone electrophoresis (CZE) under acidic as well as basic conditions. Modified peptides were synthesized for three purposes: (i) to analyze their functions in the first three acidic amino acid residues, (ii) to analyze their functions in three sequenced glycines, and (iii) to analyze the length of side chains in acidic amino acids. The roles of glycines, acidic amino acids and amino acid side chains in the binding of Mg2+ were studied at pH values less than 7.0. The migration times of GPP varied with the pH of various electrophoresis buffers, and electrophoresis patterns were significantly changed between pH 7.0–7.5. Based on the electrophoretic analysis, it was concluded that the binding mechanisms for Mg2+ or conformations of GPP changed between low pH and high pH.
Immunoblot analysis of components of barley recognized by IgE antibodies in sera from pig farm workers
Authors: Chiung Yin‐Mei , Shen Horng‐Der , Huang Ju‐Wei 
Published: May 2005

AbstractComponents of the crude extract of barley, recognized by IgE antibodies in sera from 132 pig farm‐workers in southern Taiwan, were investigated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting. Among the sera tested, 43 (32.6%) showed IgE reactivity to 15 components of barley with molecular mass ranging from 148000 to 14000 daltons (Da). Heterogeneity in the IgE‐binding patterns of the samples tested was observed. However, the major antigen appears to be the component with about 37 kDa, in 32 (74.4%) of the 43 immunoblot‐positive serum samples. Furthermore, components with about 55 and 67 kDa which reacted with 26 (60.5%) and 18 (41.9%) of the positive samples, respectively. These might also be considered as important allergens of the flour made from entire seeds of barley. The results suggest that barley may be considered to play a role in the IgE‐mediated occupational respiratory disorders and the 37 kDa component has also been shown to be the major allergen.
Two‐dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number
Authors: Wilkins Marc R. , Gasteiger Elisabeth , Sanchez Jean‐Charles , Bairoch Amos , Hochstrasser Denis F. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two‐dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2‐D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2‐D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2‐D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2‐D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2‐D gel. Considering the current maximum in 2‐D gel loading capacity, it was found that 2‐D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2‐D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.
Effects of carrier and hapten array on the production of anti‐hapten antibodies analyzed by two‐dimensional affinity electrophoresis
Authors: Wang Pei , Nakamura Kazuyuki 
Published: May 2005

AbstractEffects of a carrier, bovine serum albumin (BSA) and a hapten array, fluorescein isothiocyanate‐conjugated dextran (FITC‐DEX), on the production of anti‐FITC antibodies in BALB/c mice were analyzed by two‐dimensional affinity electrophoresis (2‐D AEP). The mice were immunized with FITC‐BSA, followed by immunizations with FITC‐BSA, saline, BSA, human serum albumin (HSA), fluorescein (Flu), and FITC‐DEX. The heterogeneity and quantity of anti‐FITC antibodies were increased markedly during the second and third immunization with FITC‐BSA. However, the production of anti‐FITC antibodies with low affinity to FITC was suppressed by the second and third immunization with the carrier protein of BSA. The suppression of anti‐FITC antibodies by immunization with BSA may be due to the major histocompatibility complex (MHC)‐mediated competition through antigen processing and the clonal expansion of BSA‐specific B cells because similar suppression was induced by the immunization with HSA. The hapten array of FITC‐DEX generally suppressed the production of anti‐FITC antibodies; however, immunization with free hapten of Flu did not affect the production significantly. This may indicate that cross‐linking of anti‐FITC antibodies on the surface of specific B cells via the binding of hapten array is required for the suppression.
Steady‐state concentration distribution of ampholytes in isoelectric focusing in a linear immobilized pH gradient
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio 
Published: May 2005

AbstractThe equation of balance between electrophoretic and diffusional mass flows in the steady state of isoelectric focusing is analyzed. To create the pH gradient, a model system composed of only one Immobiline is used. The solution is found for the case of a small sample concentration, but without assumptions about linear “focusing force” and constant (linear) conductivity profiles. The effect of sample concentration on the final concentration profile is also evaluated. At high sample concentrations, it is demonstrated that the steady‐state distribution is essentially non‐Gaussian and results in a considerably lower concentration maximum as compared with low sample levels.
On the concept of “normalized buffering power/conductivity ratio” of isoelectric buffers for capillary zone electrophoresis
Authors: Stoyanov Alexander V. , Righetti Pier Giorgio 
Published: May 2005

AbstractThe introduction of a new physico‐chemical parameter is proposed: the “normalized buffering power/conductivity ratio”. It expresses the ratio of buffering power to conductivity (Rβ/λ), normalized by the electrolyte concentration, and gives an opportunity to calculate the properties of buffering ions typically used in capillary electrophoretic separations of biopolymers. This procedure of normalization is possible due to the fact that, in the concentration range practivally used, the relationship of Rβ/λ on concentration is close to linear.