bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteome analysis of glycoforms: A review of strategies for the microcharacterisation of glycoproteins separated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Packer Nicolle H. , Pawlak Anna , Kett Warren C. , Gooley Andrew A. , Redmond John W. , Williams Keith L. 
Published: May 2005

AbstractPreparative two‐dimensional polyacrylamide electrophoresis (2‐D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2‐D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious ‘trains’ of spots which differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from post‐translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2‐D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2‐D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2‐D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2‐D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2‐D gels.
Comparing two‐dimensional electrophoretic gel images across the Internet
Authors: Lemkin Peter F. 
Published: May 2005

AbstractScientists around the world often work on similar data so the need to share results and compare data arises periodically. We describe a method of comparing two two‐dimensional (2‐D) protein gels of similar samples created in different laboratories to help identify or suggest protein spot identification. Now that 2‐D gels and associated databases frequently appear on the Internet, this opens up the possibility of visually comparing one's own experimental 2‐D gel image data with data from another gel in a remote Internet database. In general, there are a few ways to compare images: (i) slide one gel (autoradiograph or stained gel) over the other while back‐illuminated, or (ii) build at 2‐D gel computer database from both gels after scanning and analyzing these gels. These are impractical since in the first case the gel from the Internet database is not locally available. In the second, the costs of building a multi‐gel database solely to answer the question of whether a spot is the same spot may be excessive if only a single visual comparison is needed. We describe a distributed gel comparison program (URL: www‐lmmb.ncifcrf.gov/flicker) which runs on any World Wide Web (WWW) connected computer and is invoked from a Java‐capable web browser. One gel image is read from any Internet 2‐D gel database (e.g. SWISS‐2DPAGE) and the other may reside on the investigator's computer. Images may be more easily compared by first applying spatial warping or other transforms interactively on the user's computer. First, regions of interest are “landmarked” with several corresponding points in each gel image, then one gel image is warped to the geometry of the other. As the two gels are rapidly alternated, or flickered, in the same window, the user can slide one gel past the other to visually align corresponding spots by matching local morphology. This flicker‐comparison technique may be applied to analyzing other types of one‐dimensional and 2‐D biomedical images.
Two‐dimensional electrophoretic analysis of human milk‐fat‐globule membrane proteins with attention to apolipoprotein E patterns
Authors: Goldfarb Marcia 
Published: May 2005

AbstractTwo‐dimensional (2‐D; Iso Dalt method) electrophoresis was done on lipid membrane fractions from human milk samples collected in polypropylene tubes at home by the mother. The samples represent milk from two weeks to ten months post partum. Identification was made by immunoassay of Western blots. Apolipoprotein E was not easily seen on silver‐stained gels because concentration is small and spots overlap another protein. Blot assays show a more complex pattern than 34 kDa plasma apo E with molecular size isoforms at 36 kDa, 34 kDa, 20 kDa and 15 kDa. One sample was negative for apo E. Apo E plays an important role in lipid transport in tissue and in the brain. It is synthesized in a variety of tissues by specific cells. Milk may reflect several sources.
Improved electrophoretic separation and immunoblotting of beta‐amyloid (Aβ) peptides 1–40, 1–42, and 1–43
Authors: Wiltfang Jens , Smirnov Alexandr , Schnierstein Beate , Kelemen Georg , Matthies Uta , Klafki Hans‐Wolfgang , Staufenbiel Matthias , Hüther Gerald , Rüther Eckhardt , Kornhuber Johannes 
Published: May 2005

AbstractBeta‐amyloid peptides (Aβ peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble Aβ peptides, which are proteolytic fragments of the amyloid‐precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are reponsible for a small percentage of inherited AD cases (FAD) and increase the amount of Aβ peptides containing additional carboxy terminal amino acids (Aβ1–42, Aβ1–43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer Aβ peptides [5]. Aβ1–42 polymerizes more rapidly in vitro [6] than Aβ1–40 and has been identified as the major component of the brain amyloid deposits [7–9]. We recently developed a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS‐PAGE procedure, which allows the separation of Aβ1–40, Aβ1–42, and Aβ1–43 for the first time. Detection of the three Aβ peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer Aβ peptides in relation to their aggregation state.
Identification of ADP‐ribosylation factor‐6 in brush‐border membrane and early endosomes of human kidney proximal tubules
Authors: Marshansky Vladimir , Bourgoin Sylvain , Londoño Irene , Bendayan Moïse , Vinay Patrick 
Published: May 2005

AbstractThe expression and distribution of ADP‐ribosylation factor (ARF) small GTP‐binding proteins in kidney tissue was examined. Various anti‐ARF antibodies were raised against purified rec‐ARF 1 and rec‐ARF 6 and their specificity was determined. Using indirect immunofluorescence analysis of intact kidney, ARF proteins were found to be predominantly expressed in kidney tubules as compared to glomeruli. This result was further supported by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot analysis of purified human kidney glomeruli and proximal tubules. Both ARF 1 and ARF 6 were detected in purified human glomeruli and proximal tubules; however, ARF 1 was more abundant than ARF 6 in these kidney structures. Brush‐border membrane vesicles (BBMV) and early endosomes (EE) derived from the receptor‐mediated endocytosis pathway were isolated from purified proximal tubules of rat, dog and human kidney using a combination of magnesium precipitation and wheat‐germ agglutinin negative selection techniques. We demonstrated that ARF 6 is associated with BBMV and with EE derived from receptor‐mediated endocytosis pathway of human kidney proximal tubules. Using a combination of SDS‐PAGE and quantitative enhanced chemiluminescence Western blot analysis, the quantification of the ARF 6 distribution in membrane and cytoplasmic fractions of proximal tubules was made and its predominance in membrane fractions was demonstrated. By analogy with the functional role of ARF 1 in Golgi protein transport, we suggest that ARF 6 may play an important role in the regulation of receptor‐mediated endocytosis and protein reabsorption by kidney proximal tubules.
Spermatocytes and round spermatids of rat testis: The difference betweenin vivoandin vitroprotein patterns
Authors: Cossio Gabriela , Sanchez Jean‐Charles , Wettstein Rodolfo , Hochstrasser Denis F. 
Published: May 2005

AbstractDuring mammalian spermatogenesis meiotic cell division and spermiogenesis occurs. Gene expression during this process is temporally regulated at the transcriptional and translational levels but the mechanisms are not well understood. In this publication we have investigated the synthesis of proteinsin vitroto detect the proteins with a high metabolic turnover and to compare them with thein vivoprotein map. RNA of spermatocytes and round spermatid cell populations, purified by centrifugal elutriation, and total testis was isolated. The poly A+ mRNA fraction was translated using a rabbit reticulocyte lysate. The translation products were separated by two‐dimensional (2‐D) gel electrophoresis using nonlinear 3.5–10 immobilized pH gradients for the first‐dimensional separation. The gels with35S‐translated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and scanned using a phosphorimager. A highly reproducible and complex protein pattern was obtained using this methodology. Only rat testis messages were translated. Using Melanie 2 software we could compare and detect more than 1000 proteins on 2‐D radioactive images. Some changes could be observed in protein expression between the different cell types but they were not statistically significant. The comparison between the 2‐D rat testis map and thein vitrotranslated patterns show no matching between any spots. This result suggests that the post‐transcriptional modifications occurring in the reticulocyte system are not the same as those that occurin vivoin the testis. Rabbit reticulocyte proteins were detected by staining PVDF membranes with colloidal gold. Rat testis and reticulocyte patterns were completely different.
Electrophoretic analysis of the novel antigen for the gastrointestinal‐specific monoclonal antibody, A33
Authors: Ji Hong , Moritz Robert L. , Reid Gavin E. , Ritter Gerd , Catimel Bruno , Nice Ed , Heath Joan K. , White Sara J. , Welt Sydney , Old Lloyd J. , Burgess Antony W. , Simpson Richard J. 
Published: May 2005

AbstractThe murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane‐associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and > 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanised version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two‐dimensional electrophoresis (2‐DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non‐urea precast 2‐DE gels, that has facilitated the purification of the detergent (0.3% Triton X‐100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2‐DE conditions, the A33 antigen electrophoreses with an apparent Mr ˜ 41000 and pI 5.0–6.0. Attempts to isolate the A33 antigen from 2‐DE gels for direct structural analysis were unsuccessful, due to its co‐electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N‐terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N‐terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2‐DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: www.ludwig.edu.au). Under reducing 2‐DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (pI 4.6–4.8) with a single molecular weight species at Mr ˜ 55000.
Two‐dimensional electrophoresis of precision‐cut testis slices: Toxicologic application
Authors: Witzmann Frank A. , Fultz Carla D. , Wyman John F. 
Published: May 2005

AbstractAdvances in tissue slice technology and a recent novel application of this technique to reproductive toxicology using bovine testis have demonstrated the remarkable utility of this approach. The objective of the present study was to combine this in vitro toxicity test system with large‐scale two‐dimensional polyacrylamide gel electrophoresis (2‐DE) to detect and study alterations in testicular‐slice protein patterns as molecular correlates of 1,3,5‐trinitrobenzene (TNB) and 1,3‐dinitrobenzene (DNB) toxicity. Previous studies have shown that testicular slices remain viable for > 24 h and, as measured by protein synthesis inhibition, TNB causes dose‐related injury. Tissue‐slices were prepared from bovine testicles incubated for 2, 4 or 6 h and exposed to either 100 μM, 500 μM or 1 mM DNB or TNB in the incubation medium. Slices were collected, solubilized, and separated by large scale 2‐DE. Resulting protein patterns were then examined by image analysis, which revealed coefficients of variation in protein spot abundance comparable to patterns from fresh rodent tissue samples. Furthermore, specific protein alterations indicated dose‐related inductions and declines in protein abundance, some progressive over time. The results of this investigation demonstrate the potential toxicologic utility of combining in vitro tissue‐slice technology with high‐resolution 2‐DE protein mapping. The consolidation of these methods offers a novel approach for toxicity screening and testing, reduces experimental cost, and reduces the use of laboratory animals.
Effects of local changes in the helix flexibility on electrophoretic migration of DNA in agarose gel
Authors: Gisselfält Katrin , Åkerman Björn , Jonsson Mats 
Published: May 2005

AbstractWe present a study of how kinks, flexible bends, and flexible joints in the DNA helix, induced by binding cis‐diamminedichloroplatinum(II) (cis‐DDP), transdiamminedichloroplatinum(II) (trans‐DDP), and chlorodiethylenetriammineplatinum(II) (dien‐Pt) to the DNA, affect the electrophoretic migration of DNA in agarose gels. For long DNA the conformation oscillates between extended and compact states during the migration, as for native DNA. The presence of flexible joints decreases both the length of time and the step length of the cycles, but in a compensatory manner so that there is no net effect on the mobility. This demonstrates that in some cases mobility alone cannot detect pertubations in the DNA helix. Kinks and flexible bends reduce the mobility because they both lead to longer time periods of the cycles. With kinks the reduction is strongest at low fields because at high fields the kinks are straightened out; the steps thus become even longer than for native DNA. The results suggest that a combination of mobility and orientation measurements on reptating DNA can be used for distinguishing different kinds of structural alterations in the DNA.
Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single‐strand conformation analysis
Authors: Alonso Antonio , Martin Pablo , Albarran Cristina , Aguilera Beatriz , Garcia Oscar , Guzman Ana , Oliva Horacio , Sancho Manuel 
Published: May 2005

AbstractEach entire hypervariable region of the mitochondrial DNA control region was screened for mutations from paired normal and tumor DNA corresponding to a group of 21 patients (13 colorectal and 8 gastric adenocarcinomas) using both heteroduplex analysis and single‐strand conformation analysis. These two mutation scanning strategies allowed the identification of sequence alterations in 3/13 (23%) colorectal tumors and in 3/8 (37%) gastric tumors. Heteroduplex analysis showed the heteroplasmic state of the majority of these tumor mutations. Sequence analysis revealed two A:T/G:C transitions (nucleotide positions: 16241 and 16166) in hypervariable region 1 (HV1) and two C:G/T:A transitions (nucleotide positions: 76 and 312), one A:T/G:C transition (nucleotide position: 93), a 1‐basepair C:G deletion (nucleotide position: 309), and a 2‐base‐pair CC:GG insertion (nucleotide position: 309) in the HV 2 region. A considerable proportion of these mutations was found in homopolymeric regions which are highly polymorphic among humans. Different mechanisms (clonal expansion, increased oxidative damage, and nuclear mutator mutations) were suggested to explain the increased mitochondrial DNA mutation rate observed in cancer.
Quantitative description of analyte migration behavior based on dynamic complexation in capillary electrophoresis with one or more additives
Authors: Peng Xuejun , Bowser Michael T. , Britz‐McKibbin Philip , Bebault Gwendolyn M. , Morris Jamie R. , Chen David D. Y. 
Published: May 2005

AbstractA comprehensive theory is proposed to describe the migration behavior of analytes in capillary electrophoresis (CE) when one or more additives are present in the buffer solution. This theory amalgamates and extends the previous work done by others. The capacity factor (ḱ) in this theory is defined as the product of the equilibrium constant and the additive concentration, thus, ḱ changes linearly with additive concentration. The net electrophoretic mobility of an analyte is a function of ḱ, therefore, it can be changed by varying the additive concentration. Three parameters are needed to predict the mobility of an analyte in a one‐additive CE system: the mobility of the free analyte, the mobility of the complex, and the equilibrium constant for the analyte‐additive interaction (which determines the fraction of the free analyte at different additive concentrations). When additives are used, the change in viscosity obscures this relationship, therefore, a viscosity correction factor is required to convert all mobilities to an ideal state where the viscosity remains constant. The migration behavior of an analyte in a solution with multiple additives can be predicted and controlled, once the equilibrium constants of the interactions between the analyte and each of the additives are obtained separately. β‐Cyclodextrin and hydroxypropyl‐β‐cyclodextrin are used as additives and the migration behavior of phenol, p‐nitrophenol, and benzoic acid are studied as a model system to verify this theory. When the necessary viscosity correction factor is included, the net electrophoretic mobilities of the analytes obtained from experimental results agree with the values predicted by the theory based on dynamic complexation. Although only experiments with one and two additives were carried out to verify the theory, the equations apply to situations when more than two additives are used. The relationship between the theories of electrophoresis and chromatography is clarified.
Density gradient isoelectric focusing of proteins in artificial pH gradients made up of binary mixtures of amphoteric buffers
Authors: Tulp Abraham , Verwoerd Desirée , Hart Augustinus A. M. 
Published: May 2005

AbstractA density gradient electrophoresis apparatus made of Perspex (7 cm, Ø 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bieret al.(Electrophoresis1993,14, 1011–1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK1,1–1–2) and the pK2,II+1+2) region. 3‐(N‐Morpholino)propanesulfonic acid (MOPS) and γ‐amino‐n‐butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate‐zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochromec, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate‐zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycoforms of transferrin were separated and enriched on a pH 5.2–6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill‐defined Ampholines, the cost of these well‐defined amphoters is lowPresented at the “Elektrophorese Forum ”︁96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23–25, 1996.
Changes in nuclear protein composition in response to chronic electrical stimulation of skeletal muscle
Authors: Gilroy Stephen J. , Salmons Stanley , Pennington Stephen R. 
Published: May 2005

AbstractThe adaptive response of skeletal muscle to increased functional demand involves phenotypic changes that affect contractile properties, energy metabolism and calcium kinetics. Some of these changes are known to be initiated at a pre‐translational level, but the underlying regulatory mechanisms have not yet been identified. In this study we used chronic electrical stimulation (10 Hz, continuous) to initiate fast‐to‐slow muscle fibre‐type transformation, and two‐dimensional electrophoresis (2‐DE) to assess changes in nuclear protein composition after 24 and 72 h. We report an early and sustained increase in the level of a 3 kDa protein in stimulated fast muscle (n = 6). The presence of the same protein in control slow muscle is consistent with a possible functional role in the determination of the slow phenotype.
Determination of association constants of dansyl‐amino acids and β‐cyclodextrin in N‐methylformamide by capillary electrophoresis
Authors: Valkó István E. , Sirén Heli , Riekkola Marja‐Liisa 
Published: May 2005

AbstractThe use of nonaqueous background electrolytes in capillary electrophoresis (CE) is a promising new trend which should widen the scope of this technique. We demonstrate the chiral separation of dansyl‐amino acids (Dns‐AAs) in N‐methylformamide (NMF) using β‐cyclodextrin (β‐CD) as chiral selector. The solubility of β‐CD is much better in NMF than in water, allowing high concentration of the chiral selector and successful enantioseparation despite the weak host‐guest interaction between the Dns‐AAs and β‐CD. The association constants for the complexation between Dns‐AAs and β‐CD could be calculated from the electrophoretic mobilities, with attention paid to the change in viscosity of the electrolyte upon addition of β‐CD. The association constants ranged between 2 and 13 M−1.