bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of oxidative stress and Ca2+ agonists on molecular chaperones in human umbilical vein endothelial cells
Authors: Dreher Don , Vargas J. Reynaldo , Hochstrasser Denis F. , Junod Alain F. 
Published: May 2005

AbstractEndothelial cell dysfunction is a key factor in oxidative stress‐related pathology. Disruption of Ca2+ homeostasis is thought to be responsible for much of the endothelial cell dysfunction in oxidative stress. The expression of molecular chaperones (MC), which stabilize protein structures in normal and in stress conditions, reflects the Ca2+ ‐dependent and ‐independent stress effects in the different cell compartments. By two‐dimensional (2‐D) gel electrophoresis, combined with immunoblotting or microsequencing, we have identified 12 major MC in human umbilical vein endothelial cells (HUVEC): (i) the endoplasmic reticulum‐located MC GRP78, GRP94, protein disulfide isomerase, and calreticulin; (ii) the mitochondrial MC HSP65 and GRP75; and (iii) the cytosolic/nuclear MC HSP27, HSC70, HSP70, HSP90, cyclophilin, and ubiquitin. To differentiate oxidative stress‐ and Ca2+ ‐mediated effects, HUVEC were exposed to 1) xanthine oxidase plus hypoxanthine to generate oxidative stress, 2) ionomycin plus ethylene glycol‐bis(β‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA) to deplete intracellular Ca2+ stores, or 3) thrombin to increase cytosolic Ca2+. De novo protein synthesis after exposure was quantified by the incorporation of [35S]methionine. Image processing with the MELANIE system was used to create and compare the 2‐D maps of [35S]methionine‐labeled proteins under conditions 1)–3) with those of the controls. In a total of 24 2‐D gels, 9 different MC were detected in at least 5 out 6 experimental replicates and were subjected to numeric analysis. The statistics showed a > 10% increase in GRP78 (p < 0.05), HSP27, cyclophilin, and ubiquitin after oxidative stress. Ionomycin plus EGTA influenced the expression of GRP78 (p < 0.01), cyclophilin, and ubiquitin in the same direction, but in addition increased GRP94 (p < 0.01) and calreticulin. Thrombin upregulated calreticulin (p < 0.05), HSC70, and cyclophilin. In conclusion, the computer analysis of MC expression on 2‐D maps under different stress conditions reveals a differential correlation between the oxidative stress and the depletion of Ca2+ stores in HUVEC.
Analysis of proteins from human breast epithelial cells using two‐dimensional gel electrophoresis
Authors: Giometti Carol S. , Tollaksen Sandra L. , Chubb Cynthia , Williams Catherine , Huberman Eliezer 
Published: May 2005

AbstractThe human breast is a highly specialized, complex tissue comprised of a heterogeneous population of cells with varying functions. Interactions between the different cell types, changes in their relative abundance, state of differentiation and function in response to stimuli, as well as the alterations that lead to the aberrant growth associated with malignancy are poorly understood. Two‐dimensional gel electrophoresis is being used to compare the proteins found in different breast cells in order to identify the gene products that are common or specific to particular cell types so as to provide markers that will be useful in studies of normal breast cell differentiation and the dedifferentiation or blocked differentiation characteristic of cancer. Protein patterns have been obtained from cells prepared for electrophoresis immediately after isolation from human milk, from cells cultured for fewer than ten passages after isolation from healthy breast tissue removed during reduction mammoplasty, and from cells maintained in long‐term tissue culture after isolation from the pleural effusions of patients with breast carcinomas. Differential expression of cytokeratins 8, 18, and 19, shown previously to be predominantly expressed by epithelial cells in the luminal layer of breast tissue, was observed among the cells analyzed. Other non‐cytokeratin proteins were also found to be differentially expressed in subsets of both the normal and tumor cells. A composite human breast cell protein pattern was created which includes all the commonly and specifically expressed proteins found in this study. This pattern will be the basis for continuing studies of proteins in the human breast.
Repetitive treatment with serotonin modifies protein synthesis and protein phosphorylation in the central nervous system of Hirudo medicinalis
Authors: Garcia‐Gil Mercedes , Bottai Daniele , Romano Antonia , Fineschi Loredana , Bini Luca , Pallini Vitaliano , Brunelli Marcello 
Published: May 2005

AbstractSerotonin (5HT) is the neurotransmitter involved in some forms of short‐term memory in the leech. Behavioral experiments have demonstrated that long‐term memory requires new protein synthesis. With the aim of studying the molecular mechanism underlying memory processes in the leech, we have analyzed the effect of 5HT on protein synthesis and protein phosphorylation. Segmental ganglia of the leech central nervous system haves been labeled, proteins have been separated by two‐dimensional‐electrophoresis and labeled proteins detected by autoradiography. Our findings indicate that repetitive treatment with 5HT produces either the persistence of phosphorylation or changes in protein synthesis in several proteins.
Two‐dimensional electrophoresis of intracellular and secreted protein synthesized by fetal bovine chondrocytes in high‐density culture
Authors: Freyria Anne‐Marie , Ronzière Marie‐Claire , Boutillon Marguerite‐Marie , Herbage Daniel 
Published: May 2005

AbstractIn order to study the mechanisms involved in the differentiation/dedifferentiation of chondrocytes, fetal bovine chondrocytes in high‐density cultures were treated with retinoic acid, an agent known to modify the chondrocyte phenotype (10 μmol/L between day 2 to day 5 of culture). The synthesis of intracellular and secreted proteins was studied by two‐dimensional electrophoresis in cell lysates and culture media after labeling with [35S]methionine for the last 14 h of culture. The proteins expressed in control and retinoic acid‐treated cells were identified by microsequencing after “in‐gel” tryptic digestion of the spot or by immunodetection with specific antibodies after two‐dimensional gel blotting. Intracellular protein modifications included one of 56.9 kDa and with an isoelectric point (pI) of 5.8 whose synthesis was previously reported to be up‐regulated by 75%. Microsequencing of two internal peptides did not reveal a known protein. Changes to the chondrocyte phenotype were also recorded in the culture medium, as a decrease in type II collagen synthesis and expression of the small proteoglycan, decorin. Several new spots were also observed after treatment with retinoic acid, including a large, diffuse spot, not yet characterized, with a mean molecular mass of 39 kDa and a pI of 4.5–5.0. Under our experimental conditions, retinoic acid induces morphological changes of the chondrocytes and dramatic changes in the synthesis of several intracellular and secreted proteins that predate the synthesis of collagen type I (the classical marker of chondrocyte dedifferentiation).
Two‐dimensional electrophoresis of acidic proteins isolated from ozone‐stressed Norway spruce needles (Picea abies L. Karst): Separation method and image processing
Authors: Davidsen Nina B. 
Published: May 2005

AbstractThe effect of ozone stress (< 10, 200 or 400 μg m−3) on the protein pattern of Picea abies L. Karst needles was studied by fumigating potted grafts from mature trees. Clonal trees of Norway spruce were fumigated in open‐top chambers for one growing season. Proteins, soluble and membrane bound, were extracted from the needles in a four‐step procedure: (i) grinding in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenol oxidase and polyvinylpolypyrrolidone (PVP) added to bind phenolic compounds; (ii) precipitation of proteins with acetone at −20°C; (iii) solubilization in a modified lysis buffer; and (iv) dialysis against the modified lysis buffer. Proteins were separated by two‐dimensional electrophoresis according to O'Farrell's method. The changes in the silver‐stained protein pattern were evaluated by using image processing methods. Electrophoretograms from stressed and nonstressed trees are presented.
Genetic variability of carrot seed proteins analyzed by one‐ and two‐dimensional electrophoresis with immobilized pH gradients
Authors: Posch Anton , van den Berg Bartel M. , Burg Henk C. J. , Görg Angelika 
Published: May 2005

AbstractThe genetic variability of eight carrot populations representing commercially important morphological types was analyzed for seed protein polymorphism by one‐ and two‐dimensional electrophoresis using immobilized pH gradients (IPG) in order to assess the potential of both techniques in carrot hybrid breeding as well as genetic quality testing of carrot trade seeds. The digitized silverstained one‐ and two‐dimensional gels of the urea/detergent‐soluble seed proteins were subjected to computer analysis to estimate both the inter‐ and intrapopulation genetic variability. The results indicated that isoelectric focusing (IEF‐IPG) of seed proteins is a powerful tool for quick screening of the genetic variability present between and within carrot populations, while two‐dimensional polyacrylamide gel electrophoresis (IPG‐DALT) can be employed to establish detailed genetic relationships of carrot populations selected by initial IEF‐IPG screening in order to use genetic distant and homogeneous populations for successful carrot hybrid breeding. However, the applicability of IEF‐IPG in genetic quality testing of carrot seeds seems to be restricted to hybrid purity testing. IPG‐DALT is a far more powerful tool to profile unambiguously all carrot varieties commercially used in order to test the genetic quality of trade seeds, because this technique exhibits protein spots or spot combinations exclusively found in each of the eight varieties investigated.
Phosphorylation of herpes simplex virus type 1 Us11 protein is independent of viral genome expression
Authors: Simonin Denis , Diaz Jean‐Jacques , Kindbeiter Karine , Pernas Patrick , Madjar Jean‐Jacques 
Published: May 2005

AbstractThe Us11 protein is a true late gene product of herpes simplex virus type 1 (HSV‐1), whose exact function is unknown but which exhibits RNA‐binding properties and which is phosphorylated on serine residues. In order to determine whether the Us11 protein is phosphorylated by cellular kinase(s) or by virally encoded kinase(s), the Us11 gene has been cloned and transiently expressed in HeLa cells. In addition, HeLa‐derived cell lines have been selected for their ability to express Us11 protein constitutively. 32P‐Labeling and analysis by two‐dimensional electrophoresis of transiently and constitutively expressed Us11 protein demonstrated that, indeed, multiple phosphorylation of the protein occurs in absence of HSV‐1 genome expression, indicating that the protein behaves as a natural substrate for cellular kinase(s). In addition, a sequence heterogeneity of the Us11 protein, due to a difference in the number of SPREPR repeats, has been characterized between different strains of HSV‐1.
Manipulation of a single cell with microcapillary tubing based on its electrophoretic mobility
Authors: Kitagawa Shinya , Kawaura Chiyo , Hashimoto Osamu , Takahashi Tsutomu , Naoi Makoto , Tsuda Takao 
Published: May 2005

AbstractManipulation of a single cell of spherical shape, approximately 5–10 μm in diameter, was performed with capillary tubing and an electrostatic field. A single cell migrates with its electrophoretic mobility into capillary tubing against the flow of electroosmosis coming out of the capillary. After trapping the cell in the capillary, it is pulled out into the other microreservoir with the application of a reverse electric voltage. When we apply a negative voltage to the microreservoir itself, the cell in it can keep floating for a relatively long period due to electrostatic repulsion. The electrophoretic mobility of a single cell is also estimated.
Calculation of the isoelectric points of native proteins with spreading of pKa values
Authors: Henriksson Gunnar , Englund Ann‐Kristin , Johansson Gunnar , Lundahl Per 
Published: May 2005

AbstractThe isoelectric points (pI) of native proteins are important in several separation techniques. For estimating pI values the net charge of several proteins was calculated versus pH by use of the Henderson‐Hasselbalch equation. Amino acid composition, pKa values for amino acid side chains and for the N‐ and C‐terminal groups, and the presence of other charged groups were taken into account. A set of pKa values was chosen for amino acid residues with ionizable side chains. Each particular type of ionizable group was assumed to have pKa values distributed around the chosen value, thereby simulating the situation in proteins and polypeptides. The calculated pI values showed reasonably good agreement with experimental ones for most of 16 native proteins over a wide pH range (3.4–11) when charge contributions of heme groups, sialic acid residues, etc., were taken into account. The calculated pI for the human red cell glucose transporter (Glut 1) with one sialic acid residue was decreased from 8.8 to 8.5 by introducing pKa value spreading and became consistent with the experimental pI value of 8.4 ± 0.05 at 15°C determined in the presence of 6 M urea. The pI of the native Glut1 was lower, 8.0 ± 0.1, at 22°C. In general, the pI values for native proteins are affected by the three‐dimensional structure of the proteins, which causes greater differences between calculated and experimental pI values than in the case of polypeptides for which pI values are determined in the presence of urea.
Characterization of proteins by sequential isoelectric focusing on immobilized pH gradients and electrospray mass spectrometry
Authors: Breme Umberto , Breton Jerǒme , Visco Carlo , Orsini Gaetano , Righetti Pier Giorgio 
Published: May 2005

AbstractThe coupling of isoelectric focusing on immobilized pH gradients (IPG) with electrospray‐mass spectrometry (ES‐MS) was applied to the characterization of proteins according to two different and important properties, such as net surface charge and molecular mass. From a technical point of view, these methods are complementary, since ES‐MS requires ion‐free samples as usually supplied by isoelectric focusing on IPGs. This report describes the experiments carried out on model proteins to demonstrate the feasibility of the sequential application of these two techniques for the characterization of proteins. A minimum of 5 μg protein was needed for good signal by mass spectrometry. The following proteins were studied: myoglobin, truncated interleukin‐6‐mutein, recombinant cytochrome c551 and insulin‐like growth factor I. Extraction from the IPG matrix was carried out in 70% acetonitrile/30% water/0.05% trifluoroacetic acid either by passive diffusion or by centrifugation through a 0.22 μm Amicon membrane, with protein recoveries of 80–85%.
DNA separation with field inversion capillary electrophoresis
Authors: Heller Christoph , Pakleza Christophe , Viovy Jean Louis 
Published: May 2005

AbstractWe have built an automated pulsed field capillary electrophoresis system on the basis of a commercially available device. Using entangled polymer solutions as separating matrix, we demonstrate a considerable improvement of separation of double‐stranded DNA in the range of 1–50 kbp under pulsed field conditions. The influence of the main parameters, i.e. pulse frequency and electric field, is studied and the results are compared to existing electrophoresis theories.
Gene linkage of two‐dimensional polyacrylamide gel electrophoresis resolved proteins from isogene families in Saccharomyces cerevisiae by microsequencing of in‐gel trypsin generated peptides
Authors: Norbeck Joakim , Blomberg Anders 
Published: May 2005

AbstractThe total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) where 500 μg was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis. Peptides were generated from resolved proteins by in‐gel trypsin digestion, and fractionated by reversed phase‐high performance liquid chromatography (RP‐HPLC). Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PDC1, ENO1 ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2. The chromatograms obtained from RP‐HPLC of related proteins were utilized to distinguish discriminating peptide fractions. With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated. We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2‐D pattern of S. cerevisiae will be obtained in sequencable amounts.
An new mode of size‐dependent separation of proteins by capillary electrophoresis in presence of sodium dodecyl sulfate and concentrated oligomeric dextran
Authors: Takagi Toshio , Karim Mohammad Rezaul 
Published: May 2005

AbstractCapillary electrophoresis in presence of sodium dodecyl sulfate (SDS) and 10% w/v of oligomeric dextran with molecular weight of just above 1000 was found to be an efficient medium for size‐dependent separation of proteins (Karim, M. R., Janson, J.‐C., Takagi, T., Electrophoresis 1994, 15, 1531–1534). The separations were found to be highly reproducible, both with respect to the position and relative amounts of the peaks. No evidence for a network structure in the medium could be found on examination of the effect of dextran concentration and viscosity measurements. The separation mechanism appears to be different from sieving by a polymer network structure. In free solution, SDS‐protein complexes with molecular weights > 10000 migrate with almost the same mobilities, irrespective of their molecular weight (this behavior is called free draining). Such SDS‐complexes of proteins or peptides with molecular weights < 10000, however, tend to increase their negatively signed mobilities with decreasing molecular weight (Karim, M. R., Shinagawa, S., Takagi, T., Electrophoresis 1994, 15, 1141–1146). Our working hypothesis is that in presence of the oligomeric dextran, the range of deviation from the free‐draining electrophoretic behavior extends to higher molecular weights, resulting in a novel separation made.
Sodium dodecyl sulfate‐gel electrophoresis of proteins employing short capillaries
Authors: Manabe Takashi 
Published: May 2005

AbstractThe performance of capillary gel‐sodium dodecyl sulfate (SDS) electrophoresis for molecular‐mass‐dependent separation of proteins has been examined employing cross‐linked polyacrylamide gels and untreated fused‐silica capillaries of short length. An apparatus for capillary electrophoresis has been constructed, combining a UV detector equipped with a capillary holder, a power supply, and a micromanipulator for sample loading. The apparatus is capable of accomodating gel capillaries of different lengths and can be operated with two methods of sample loading: manual injection on top of the gel or electrokinetic injection. Standard proteins have been separated according to their molecular mass (ranging from 17000 to 116000) within 30 min, employing gel capillaries of effective lengths shorter than 10 cm and polyacrylamide gels ranging from 2.4 to 4.8%T (5%C). The results confirmed that gel capillaries of effective length less than 10 cm can be used for protein size‐separation by SDS electrophoresis, with much higher performance than is achieved in rod gel‐SDS electrophoresis.
The use of capillary electrophoresis for the determination of hemoglobin variants
Authors: Castagnola Massimo , Messana Irene , Cassiano Loredana , Rabino Rita , Rossetti Diana V. , Giardina Bruno 
Published: May 2005

AbstractThe application of capillary electrophoresis and related techniques for the detection of hemoglobin variants is described. Capillary zone electrophoresis (CZE) was applied for the analysis of intact tetrameric hemoglobin. CZE under denaturing conditions was used for the separation of globin chains. Both CZE and micellar electrokinetic capillary chromatography were applied for a fast and sensitive separation of tryptic digests and for the analysis of amino acid derivatives.
Differential expression of nucleophosmin and stathmin in human T lymphoblastic cell lines, CCRF‐CEM and JURKAT analyzed by two‐dimensional gel electrophoresis
Authors: Nakamura Kazuyuki , Fujimoto Masanori , Tanaka Tatehiko , Fujikura Yoshihisa 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis was used to study the expression of intracellular proteins in adherent cells of human T lymphoblastic cell line, CCRF‐CEM. The adherent cells grown in monolayer on a culture plate decreased the amount of proteins of Mr 37 000 and pI 4.7–4.9, and of 17 000 and pI 5.7. The proteins were identified to be nucleophosmin for the 37 000 protein and stathmin for the 17 000 protein by microsequencing their CNBr fragments. The amount of proteins was increased in CCRF‐CEM cells grown in floating mass to a comparable level of JURKAT cells which grew in floating mass throughout the culture. The adherent cells decreased their growth rate as compared with the cells in the floating mass. These results suggest that the adhesion of human T lymphoblastic cells modulates their morphology and proliferation via a concomitant decrease in the amount of nucleophosmin and stathmin.
Biological significance of minisatellites
Authors: Singh Lalji 
Published: May 2005

AbstractMinisatellites are tandemly repeated, highly variable DNA sequences found in most higher eukaryotes. These contain a core sequence resembling the chi sequence of Escherichia coli, which is a binding site for recombination proteins. Based on this, a generalized function of minisatellites to provide binding sites for recombination proteins in eukaryotes has also been suggested. However, recent discoveries of trinucleotide repeat expansion mutations associated with at least four human genetic diseases, several short repeats acting as motifs for binding of various transcription factors, and several minisatellite‐binding nuclear proteins, which are expressed in specific tissues and bind to specific sequences, strongly suggest that different families of minisatellites may have different functions. A banded krait minor (Bkm) satellite DNA, consisting of highly conserved GATA repeats, which is arranged in a sex‐specific manner, is hypervariable. We have found a sex‐ and tissue‐specific factor designated as Bkm‐binding protein (BBP), which specifically binds to Bkm (GATA), in the germ cells of the heterogametic sex [ovary, in the case of female heterogamety (in snakes); and testis, in the case of male heterogamety (in mice, rats and humans)]. It is in these tissues that decondensation of the W and Y chromosomes occurs. We suggest that GATA repeats of Bkm bring about a coordinated decondensation of the W and Y sex chromosomes in the germ cells in response to BBP, which may serve as a “switch” for the activation of the genes present on the W and Y sex chromosomes. Since the number of GATA repeats, in tandem, necessary for the binding of BBP is flexible, there is no selection pressure on the maintenance of the exact length of the repeats, unless it is reduced below a threshold at which the binding is completely abolished.